Robert L. Satcher

Bone Regeneration Mediated by Biomimetic Mineralization of a Nanofiber Matrix

Rapid bone regeneration within a three-dimensional defect without the use of bone grafts, exogenous growth factors, or cells remains a major challenge. We report here on the use of self-assembling peptide nanostructured gels to promote bone regeneration that have the capacity to mineralize in biomimetic fashion. The main molecular design was the use of phosphoserine residues in the sequence of a peptide amphiphile known to nucleate hydroxyapatite crystals on the surfaces of nanofibers. We tested the system in a rat femoral critical-size defect by placing pre-assembled nanofiber gels in a 5mm gap and analyzed bone formation with micro-computed tomography and histology. We found within 4 weeks significantly higher bone formation relative to controls lacking phosphorylated residues and comparable bone formation to that observed in animals treated with a clinically used allogenic bone matrix.

BMP4 Promotes Prostate Tumor Growth in Bone Through Osteogenesis

Induction of new bone formation is frequently seen in the bone lesions from prostate cancer. However, whether osteogenesis is necessary for prostate tumor growth in bone is unknown. Recently, 2 xenografts, MDA-PCa-118b and MDA-PCa-133, were generated from prostate cancer bone metastases. When implanted subcutaneously in severe combined immunodeficient (SCID) mice, MDA-PCa-118b induced strong ectopic bone formation while MDA-PCa-133 did not. To identify the factors that are involved in bone formation, we compared the expression of secreted factors (secretome) from MDA-PCa-118b and MDA-PCa-133 by cytokine array. We found that the osteogenic MDA-PCa-118b xenograft expressed higher levels of bone morphogenetic protein BMP4 and several cytokines including interleukin-8, growth-related protein (GRO), and CCL2. We showed that BMP4 secreted from MDA-PCa-118b contributed to about a third of the osteogenic differentiation seen in MDA-PCa-118b tumors. The conditioned media from MDA-PCa-118b induced a higher level of osteoblast differentiation, which was significantly reduced by treatment with BMP4 neutralizing antibody or the small molecule BMP receptor 1 inhibitor LDN-193189. BMP4 did not elicit an autocrine effect on MDA-PCa-118b, which expressed low to undetectable levels of BMP receptors. Treatment of SCID mice bearing MDA-PCa-118b tumors with LDN-193189 significantly reduced tumor growth. Thus, these studies support a role of BMP4-mediated osteogenesis in the progression of prostate cancer in bone.

Targeting prostate cancer angiogenesis through metastasis-associated protein 1 (MTA1)

Metastasis‐associated protein 1 (MTA1) is overexpressed in many forms of cancer types but its role in prostate cancer (PCa) progression and metastasis has not been explored. In this study, we addressed the functional and biological role of MTA1 in PCa.

MCT-1 Oncogene Contributes to Increased In vivo Tumorigenicity of MCF7 Cells by Promotion of Angiogenesis and Inhibition of Apoptosis

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.

Identification of estrogen-responsive genes involved in breast cancer metastases to the bone

Bone metastasis is the most common metastasis in breast cancer patients. Clinical observations propose strong association between estrogen receptor (ER)-positive tumors and the development of bone metastases. We hypothesized of biologically diverse sets of hormone-dependent tumors predisposed to bone metastases and of possible role of ER-signaling pathways in the development and progression of bone metastases. We developed a novel in vitro estrogen (E2)-responsive model system, in which breast cancer cells and bone cells express high levels of either ERα or ERβ. Using co-culture approach and gene array technology we identified E2-responsive genes involved in the interaction between cancer cells and bone cells. We detected 13 genes that were altered solely by ERα and 11 genes that were regulated solely by ERβ in cancer cells. Only 5 genes were modified by both ERα and ERβ. Interestingly, the majority of genes in bone cells were altered through ERβ. Two genes, namely MacMarcks and Muc-1, whose changes in expressions in cancer cells in response to E2 were highly significant, were selected for immunohistochemical analysis using tissue microarrays of 59 infiltrating ductal carcinomas. Our results indicated that both MacMarcks and Muc-1 were expressed at high frequency in ER-positive tumors. The correlation between ERα- and ERβ-status of hormone-dependent tumors with combined expression of these two markers might suggest a more aggressive tumor phenotype associated with bone metastases. Further analysis of tissues with clinicopathological characteristics and known bone metastatic disease will indicate potential prognostic values of these and other markers in the development of bone metastases in a subgroup of “bad” hormone-dependent breast cancer.

Inhibition of Cell Adhesion by a Cadherin-11 Antibody Thwarts Bone Metastasis

Cadherin-11 (CDH11) is a member of the cadherin superfamily mainly expressed in osteoblasts but not in epithelial cells. However, prostate cancer cells with a propensity for bone metastasis express high levels of cadherin-11 and reduced levels of E-cadherin. Downregulation of cadherin-11 inhibits interaction of prostate cancer cells with osteoblasts in vitro and homing of prostate cancer cells to bone in an animal model of metastasis. These findings indicate that targeting cadherin-11 may prevent prostate cancer bone metastasis. To explore this possibility, a panel of 21 monoclonal antibodies (mAb) was generated against the extracellular (EC) domain of cadherin-11. Two antibodies, mAbs 2C7 and 1A5, inhibited cadherin-11–mediated cell–cell aggregation in vitro using L-cells transfected with cadherin-11. Both antibodies demonstrated specificity to cadherin-11, and neither antibody recognized E-cadherin or N-cadherin on C4-2B or PC3 cells, respectively. Furthermore, mAb 2C7 inhibited cadherin-11–mediated aggregation between the highly metastatic PC3-mm2 cells and MC3T3-E1 osteoblasts. Mechanistically, a series of deletion mutants revealed a unique motif, aa 343-348, in the cadherin-11 EC3 domain that is recognized by mAb 2C7 and that this motif coordinated cell–cell adhesion. Importantly, administration of mAb 2C7 in a prophylactic setting effectively prevented metastasis of PC3-mm2 cells to bone in an in vivo mouse model. These results show that targeting the extracellular domain of cadherin-11 can limit cellular adhesion and metastatic dissemination of prostate cancer cells. Implications: Monotherapy using a cadherin-11 antibody is a suitable option for the prevention of bone metastases. Mol Cancer Res; 11(11); 1401–11. ©2013 AACR.

Modulation of prostate cancer cell gene expression by cell-to-cell contact with bone marrow stromal cells or osteoblasts

After prostate cancer cells (PCa) arrive in bone, interactions with cells that include long bone osteoblasts (LBOB) and bone marrow stromal cells (BMSC) lead to metastasis formation. The effect of heterotypic cell–cell contact between PCa cells and BMSC or LBOB on PCa cell gene expression is poorly understood. To establish the role of heterotypic contact in bone metastasis formation, we mixed and co-cultured PC3 cells with rat BMSC, LBOB, or human prostate stromal cells (PS15). PC3 cells were then re-isolated for gene array analysis, and imaged using in situ hybridization to confirm that heterotypic contact regulates gene expression. The gene expression was examined using focused gene arrays containing 96 each of tumor metastasis genes or osteogenesis genes. A total of 18 out of 192 genes in PC3 cells were found to be under or over expressed subsequent to heterotypic contact with BMSC when analyzed. A total of 15 genes out of 192 were regulated in co-culture with LBOB, and 19 genes with PS15. Only two genes, uPA and Collagen III, were regulated by contact with BMSC or LBOB (both are bone derived cells), but not by contact with PS15. The relationship between cell–cell contact and uPA expression was further explored by varying cell ratios in co-culture. uPA over-expression in PC3 was related to the BMSC:PC3 ratio, and was maximum at a 10:1 ratio, where most PC3 cells would be in contact with BMSC, as predicted by a theoretical model of heterotypic contact. In situ staining of micropatterned PC3 and BMSC cells showed that uPA over-expression localizes to regions of heterotypic cell–cell contact. Taken together, our results suggest that heterotypic cell-to-cell contact between PC3 and BMSC proportionally enhances gene expression for uPA, providing a mechanism for localized control of invasiveness.

Bone cancer, version 2.2017 featured updates to the NCCN guidelines

The NCCN Guidelines for Bone Cancer provide interdisciplinary recommendations for treating chordoma, chondrosarcoma, giant cell tumor of bone, Ewing sarcoma, and osteosarcoma. These NCCN Guidelines Insights summarize the NCCN Bone Cancer Panels guideline recommendations for treating Ewing sarcoma. The data underlying these treatment recommendations are also discussed.

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.

Three-dimensional (3D) culture of bone-derived human 786-O renal cell carcinoma retains relevant clinical characteristics of bone metastases

Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems.