ERß1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer.

BACKGROUND: The role and clinical value of ERß1 expression is controversial and recent data demonstrates that many ERß antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERß1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression. METHODS: Nuclear and cytoplasmic expression patterns of ERß1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERß1, we generated multiple ERß1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERß-specific agonist exposure. RESULTS: Nuclear ERß1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERß1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERß-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective. CONCLUSIONS: Using a validated antibody, we have confirmed that nuclear ERß1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERß appears to be a novel therapeutic target. However, the efficacy of SERMs and ERß-specific agonists differ as a function of ERα expression.

The effects of a novel hormonal breast cancer therapy, endoxifen, on the mouse skeleton.

Endoxifen has recently been identified as the predominant active metabolite of tamoxifen and is currently being developed as a novel hormonal therapy for the treatment of endocrine sensitive breast cancer. Based on past studies in breast cancer cells and model systems, endoxifen classically functions as an anti-estrogenic compound. Since estrogen and estrogen receptors play critical roles in mediating bone homeostasis, and endoxifen is currently being implemented as a novel breast cancer therapy, we sought to comprehensively characterize the in vivo effects of endoxifen on the mouse skeleton. Two month old ovariectomized C57BL/6 mice were treated with vehicle or 50 mg/kg/day endoxifen hydrochloride via oral gavage for 45 days. Animals were analyzed by dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, micro-computed tomography and histomorphometry. Serum from control and endoxifen treated mice was evaluated for bone resorption and bone formation markers. Gene expression changes were monitored in osteoblasts, osteoclasts and the cortical shells of long bones from endoxifen treated mice and in a human fetal osteoblast cell line. Endoxifen treatment led to significantly higher bone mineral density and bone mineral content throughout the skeleton relative to control animals. Endoxifen treatment also resulted in increased numbers of osteoblasts and osteoclasts per tissue area, which was corroborated by increased serum levels of bone formation and resorption markers. Finally, endoxifen induced the expression of osteoblast, osteoclast and osteocyte marker genes. These studies are the first to examine the in vivo and in vitro impacts of endoxifen on bone and our results demonstrate that endoxifen increases cancellous as well as cortical bone mass in ovariectomized mice, effects that may have implications for postmenopausal breast cancer patients.

TGFß inducible early gene-1 plays an important role in mediating estrogen signaling in the skeleton.

TGFß Inducible Early Gene-1 (TIEG1) knockout (KO) mice display a sex-specific osteopenic phenotype characterized by low bone mineral density, bone mineral content, and overall loss of bone strength in female mice. We, therefore, speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. To test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell, and biochemical level. Dual-energy X-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro-CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild-type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter and resorptive activity as determined by serum levels of CTX-1 were differentially regulated after estrogen treatment in TIEG1 KO mice compared with wild-type littermates. No significant differences were detected in serum levels of P1NP between wild-type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the sex-specific osteopenic phenotype observed in female TIEG1 KO mice.

Endoxifen's molecular mechanisms of action are concentration dependent and different than that of other anti-estrogens.

Endoxifen, a cytochrome P450 mediated tamoxifen metabolite, is being developed as a drug for the treatment of estrogen receptor (ER) positive breast cancer. Endoxifen is known to be a potent anti-estrogen and its mechanisms of action are still being elucidated. Here, we demonstrate that endoxifen-mediated recruitment of ERα to known target genes differs from that of 4-hydroxy-tamoxifen (4HT) and ICI-182,780 (ICI). Global gene expression profiling of MCF7 cells revealed substantial differences in the transcriptome following treatment with 4HT, endoxifen and ICI, both in the presence and absence of estrogen. Alterations in endoxifen concentrations also dramatically altered the gene expression profiles of MCF7 cells, even in the presence of clinically relevant concentrations of tamoxifen and its metabolites, 4HT and N-desmethyl-tamoxifen (NDT). Pathway analysis of differentially regulated genes revealed substantial differences related to endoxifen concentrations including significant induction of cell cycle arrest and markers of apoptosis following treatment with high, but not low, concentrations of endoxifen. Taken together, these data demonstrate that endoxifen's mechanism of action is different from that of 4HT and ICI and provide mechanistic insight into the potential importance of endoxifen in the suppression of breast cancer growth and progression.

Klf10 inhibits IL-12p40 production in macrophage colony-stimulating factor-induced mouse bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMMs) treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), differentiate into GM-CSF-induced mouse bone marrow-derived macrophages (GM-BMMs) or M-CSF-induced mouse bone marrow-derived macrophages (M-BMMs), which have an M1 or M2 profile, respectively. GM-BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M-BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M-BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL-12(low) IL-10(high) phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel-like transcription factors and was initially described as a TGF-ß inducible early gene 1. IL-12p40 is upregulated in LPS-stimulated M-BMMs from Klf10-deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel-like factor family, can also repress the production of IL-12p40. Furthermore, Klf10 binds to the CACCC element of the IL-12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL-12p40 in M-BMMs.

TGFß-inducible early gene-1 (TIEG1) mutations in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease. A recent study showed that male KLF10-encoded TGFß Inducible Early Gene-1 knock-out mice (TIEG-/-) develop HCM with 13-fold up-regulation of PTTG1-encoded pituitary tumor-transforming gene 1. We hypothesized TIEG1 could be a novel candidate gene in the pathogenesis of genotype negative HCM in humans, possibly through a loss of its repression on PTTG1 expression. A cohort of 923 unrelated patients from two independent HCM centers was analyzed for mutations in TIEG's four translated exons using DHPLC and direct DNA-sequencing. Site directed mutagenesis was performed to clone novel variants. The effect of TIEG1 mutations on SMAD7 and PTTG1 promoters was studied using transient transfection and luciferase-assays. Altered expression of PTTG1 in cardiac tissue was studied by immunohistochemistry (IHC) to determine levels of PTTG1 protein in hypertrophic diseases. Six novel TIEG1 missense mutations were discovered in six patients (two males/four females, mean age at diagnosis 56.2±23 years, MLVWT 20.8±4 mm). Compared to WT TIEG1, five TIEG1 mutants significantly increased PTTG1 promoter function similar to TIEG1-/--mice. By IHC, PTTG1-protein expression was significantly increased in multiple models of hypertrophic cardiac disease, including TIEG1-mutation positive HCM compared to normal hearts. This is the first article to associate mutations in TIEG1 to human disease with the discovery of six novel, HCM-associated variants. Functional assays suggest a role for PTTG1 in the pathogenesis of TIEG1-mediated HCM. Up-regulation of PTTG1 seems to be a common pathway in hypertrophic heart disease, including TIEG1-mediated HCM.

Development, characterization, and applications of a novel estrogen receptor beta monoclonal antibody.

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERß and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERß and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERß antibodies are insensitive for ERß and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERß antibody is shown to be highly specific and sensitive for detection of full-length ERß and its variant forms. Strong and variable staining patterns for endogenous levels of ERß protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERß was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERß antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERß in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERß, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.

TGF-ß1 signaling and Krüppel-like factor 10 regulate bone marrow-derived proangiogenic cell differentiation, function, and neovascularization.

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-ß1-responsive Krüppel- like factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ∼ 60-fold higher than in progenitors lacking PAC markers. KLF10(-/-) mice present with marked defects in PAC differentiation, function, TGF-ß responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10(-/-) PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-ß1(+/-) CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-ß1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states.

TIEG1/KLF10 modulates Runx2 expression and activity in osteoblasts.

Deletion of TIEG1/KLF10 in mice results in a gender specific osteopenic skeletal phenotype with significant defects in both cortical and trabecular bone, which are observed only in female animals. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display reduced expression levels of multiple bone related genes, including Runx2, and exhibit significant delays in their mineralization rates relative to wildtype controls. These data suggest that TIEG1 plays an important role in regulating Runx2 expression in bone and that decreased Runx2 expression in TIEG1 KO mice is in part responsible for the observed osteopenic phenotype. In this manuscript, data is presented demonstrating that over-expression of TIEG1 results in increased expression of Runx2 while repression of TIEG1 results in suppression of Runx2. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Runx2 promoter. The zinc finger containing domain of TIEG1 is necessary for this regulation supporting that activation occurs through direct DNA binding. A role for the ubiquitin/proteasome pathway in fine tuning the regulation of Runx2 expression by TIEG1 is also implicated in this study. Additionally, the regulation of Runx2 expression by cytokines such as TGFß1 and BMP2 is shown to be inhibited in the absence of TIEG1. Co-immunoprecipitation and co-localization assays indicate that TIEG1 protein associates with Runx2 protein resulting in co-activation of Runx2 transcriptional activity. Lastly, Runx2 adenoviral infection of TIEG1 KO calvarial osteoblasts leads to increased expression of Runx2 and enhancement of their ability to differentiate and mineralize in culture. Taken together, these data implicate an important role for TIEG1 in regulating the expression and activity of Runx2 in osteoblasts and suggest that decreased expression of Runx2 in TIEG1 KO mice contributes to the observed osteopenic bone phenotype.

Noncanonical K27-linked polyubiquitination of TIEG1 regulates Foxp3 expression and tumor growth.

Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-ß-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-ß-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.

Estrogen receptor-beta sensitizes breast cancer cells to the anti-estrogenic actions of endoxifen.

INTRODUCTION: We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERα). However, the relevance of ERß in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERß and examine its effectiveness as an anti-estrogenic agent in these cell lines. METHODS: MCF7, Hs578T and U2OS cells were stably transfected with full-length ERß. ERß protein stability, dimer formation with ERα and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERß-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes. RESULTS: Our data demonstrate that endoxifen stabilizes ERß protein, unlike its targeted degradation of ERα, and induces ERα/ERß heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERß is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20 to 40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERß, whereas much higher endoxifen concentrations are needed when ERß is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40 nM) when comparing MCF7 cells which express ERß to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations. CONCLUSIONS: Taken together, these data demonstrate that the presence of ERß enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERα/ß heterodimers. These findings underscore the need to further elucidate the role of ERß in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERß expression.

TGF-ß inducible early gene 1 regulates osteoclast differentiation and survival by mediating the NFATc1, AKT, and MEK/ERK signaling pathways.

TGF-ß Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-ß treatment. As reported previously, TIEG1(-/-) mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1(-/-) osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1(-/-) precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1(-/-) osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1(-/-) osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1(-/-) cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1(-/-) precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.

Histone demethylase JARID1B/KDM5B is a corepressor of TIEG1/KLF10.

JARID1B/KDM5B (jumonji AT-rich interactive domain 1B/lysine-specific demethylase 5B) is an enzyme that efficiently removes methyl residues from trimethylated lysine 4 on histone H3, a pivotal mark for active chromatin. TIEG1/KLF10 (transforming growth factor-ß inducible early gene-1/Krüppel-like transcription factor 10) is a zinc-finger transcription factor that is involved in bone metabolism and exerts antiproliferative activity. Here, we found that TIEG1 interacts with JARID1B. In particular, the repression domains of TIEG1 bind to the C-terminus of JARID1B. Moreover, overexpression of JARID1B augments TIEG1 to repress transcription of Smad7, an inhibitor of the TGF-ß (transforming growth factor-ß) signaling pathway. Conversely, JARID1B knock-down leads to increased Smad7 mRNA levels. Thus, TIEG1 and JARID1B may cooperate to suppress tumorigenesis by enhancing TGF-ß signaling. Notably, both TIEG1 and JARID1B are downregulated in melanomas, suggesting that they indeed cooperate physiologically. In conclusion, JARID1B is the first TIEG1 corepressor identified, explaining how TIEG1 represses transcription through inducing histone H3 lysine 4 demethylation, which may be important for TIEG1 function in both normal and cancer cells.

Retinoblastoma binding protein-1 (RBP1) is a Runx2 coactivator and promotes osteoblastic differentiation.

BACKGROUND: Numerous transcription factors are involved in the establishment and maintenance of the osteoblastic phenotype, such as Runx2, osterix and Dlx5. The transcription factor retinoblastoma binding protein-1 (RBP1) was recently identified as an estrogen regulated gene in an osteosarcoma cell model. Since the function of RBP1 in osteoblastic differentiation and mineralization is unknown, we investigated the role of RBP1 in these processes. METHODS: To create a cell model with suppressed RBP1 expression, primary calvarial osteoblasts were infected with a shRNA lentiviral vector specific for mouse RBP1 (CalOB-DeltaRBP1) or a scrambled control shRNA lentivirus (CalOB-Control). Stable cell lines were generated and their mineralization potential was determined using osteoblastic differentiation medium, Alizarin Red staining, and quantitative PCR (QPCR) analyses. Runx2 coactivation by RBP1 was determined through the use of transient transfection assays. RESULTS: Stable expression of the RBP1 shRNA lentivirus in CalOB-DeltaRBP1 cells resulted in a 65-70% suppression of RBP1 expression. Osteoblastic mineralization assays demonstrated that suppression of RBP1 results in a potent delay in osteoblastic nodule formation in the CalOB-DeltaRBP1 cells with a concomitant decrease in the expression of the osteogenic transcription factors Runx2 and osterix, along with decreases in BMP2, alkaline phosphatase, osteocalcin and bone sialoprotein. Regulation of Runx2 expression by RBP1 was shown to be mediated through the proximal P2 Runx2 promoter. Furthermore, RBP1 was demonstrated to be a potent coactivator of Runx2 transcriptional activity on two known Runx2 reporter constructs. These data suggest that the expression and activity of Runx2 is critically dependent on the presence of RBP1. CONCLUSIONS: This study is the first to demonstrate that RBP1 is an important mediator of the osteoblastic phenotype and clearly defines RBP1 as a novel transcription factor involved in the well known Runx2-osterix transcriptional cascade. As such, the effects of RBP1 on these processes are mediated through both regulation of Runx2 expression and transcriptional activity.

Coprescription of tamoxifen and medications that inhibit CYP2D6.

Evidence has emerged that the clinical benefit of tamoxifen is related to the functional status of the hepatic metabolizing enzyme cytochrome P450 2D6 (CYP2D6). CYP2D6 is the key enzyme responsible for the generation of the potent tamoxifen metabolite, endoxifen. Multiple studies have examined the relationship of CYP2D6 status to breast cancer outcomes in tamoxifen-treated women; the majority of studies demonstrated that women with impaired CYP2D6 metabolism have lower endoxifen concentrations and a greater risk of breast cancer recurrence. As a result, practitioners must be aware that some of the most commonly prescribed medications coadministered with tamoxifen interfere with CYP2D6 function, thereby reducing endoxifen concentrations and potentially increasing the risk of breast cancer recurrence. After reviewing the published data regarding tamoxifen metabolism and the evidence relating CYP2D6 status to breast cancer outcomes in tamoxifen-treated patients, we are providing a guide for the use of medications that inhibit CYP2D6 in patients administered tamoxifen.

A promising approach for treatment of tumor-induced bone diseases: utilizing bisphosphonate derivatives of nucleoside antimetabolites.

Despite palliative treatments, tumor-induced bone disease (TIBD) remains highly debilitating for many cancer patients and progression typically results in death within two years. Therefore, more effective therapies with enhanced anti-resorptive and cytotoxic characteristics are needed. We developed bisphosphonate-chemotherapeutic conjugates designed to bind bone and hydrolyze, releasing both compounds, thereby targeting both osteoclasts and tumor cells. This study examined the effects of our lead compound, MBC-11 (the anhydride formed between arabinocytidine (AraC)-5'-phosphate and etidronate), on bone tumor burden, bone volume, femur bone mineral density (BMD), and overall survival using two distinct mouse models of TIBD, the 4T1/luc breast cancer and the KAS-6/1-MIP1alpha multiple myeloma models. In mice orthotopically inoculated with 4T1/luc mouse mammary cells, MBC-11 (0.04 microg/day; s.c.) reduced the incidence of bone metastases to 40% (4/10), compared to 90% (9/10; p=0.057) and 100% (5/5; p=0.04) of PBS- or similarly-dosed, zoledronate-treated mice, respectively. MBC-11 also significantly decreased bone tumor burden compared to PBS- or zoledronate-treated mice (p=0.021, p=0.017, respectively). MBC-11 and zoledronate (0.04 microg/day) significantly increased bone volume by two- and four-fold, respectively, compared to PBS-treated mice (p=0.005, p<0.001, respectively). In mice systemically injected with human multiple myeloma KAS-6/1-MIP1alpha cells, 0.04 and 4.0 microg/day MBC-11 improved femur BMD by 13% and 16%, respectively, compared to PBS (p=0.025, p=0.017, respectively) at 10 weeks post-tumor cell injection and increased mean survival to 95 days compared to 77 days in mice treated with PBS (p=0.047). Similar doses of zoledronate also improved femur BMD (p< or =0.01 vs PBS) and increased mean survival to 86 days, but this was not significantly different than in PBS-treated mice (p=0.53). These results demonstrate that MBC-11 decreases bone tumor burden, maintains bone structure, and may increase overall survival, warranting further investigation as a treatment for TIBD.

Functional role of KLF10 in multiple disease processes.

Since the discovery by this laboratory of the zinc finger transcription factor, KLF10, a member of the Krüppel-like family of transcription factors, there have been multiple publications regarding its functions and its immediate family members, in numerous cell types. KLF10 has been shown to be rapidly induced by TGFbeta1, 2, 3, E(2), epidermal growth factor, and bone morphogenetic protein-2. TGFbeta inducible early gene-1 activates the TGFbeta-Smad signaling pathway via repression of Smad 7 expression and activation of Smad 2 expression and activity. Overall, KLF10 has been implicated in cell differentiation, as a target gene for a variety of signaling pathways, and in serving as a potential marker for human diseases such as breast cancer, cardiac hypertrophy, and osteoporosis. Like other KLF members, KLF10 is expressed in specific cell types in numerous tissues and is known to be involved in repressing cell proliferation and inflammation as well as inducing apoptosis similar to that of TGFbeta. KLF10 binds to Sp-1-GC rich DNA sequences and can activate or repress the transcription of a number of genes. Overall, KLF10 has been shown to play a major role in the TGFbeta inhibition of cell proliferation and inflammation and induction of apoptosis, and its overexpression in human osteoblasts and pancreatic carcinoma cells mimics the actions of TGFbeta.

2-methoxyestradiol-mediated anti-tumor effect increases osteoprotegerin expression in osteosarcoma cells.

Osteosarcoma is a bone tumor that frequently develops during adolescence. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, induces cell cycle arrest and cell death in human osteosarcoma cells. To investigate whether the osteoprotegrin (OPG) protein plays a role in 2-ME actions, we studied the effect of 2-ME treatment on OPG gene expression in human osteosarcoma cells. 2-ME treatment induced OPG gene promoter activity and mRNA levels. Also, Western blot analysis showed that 2-ME treatment increased OPG protein levels in MG63, KHOS, 143B and LM7 osteosarcoma cells by 3-, 1.9-, 2.8-, and 2.5-fold, respectively, but did not affect OPG expression in normal bone cells. In addition, increases in OPG protein levels were observed in osteosarcoma cell culture media after 3 days of 2-ME treatment. The effect of 2-ME on osteosarcoma cells was ligand-specific as parent estrogen, 17beta-estradiol and a tumorigenic estrogen metabolite, 16alpha-hydroxyestradiol, which do not affect osteosarcoma cell cycle and cell death, had no effect on OPG protein expression. Furthermore, co-treating osteosarcoma cells with OPG protein did not further enhance 2-ME-mediated anti-tumor effects. OPG-released in 2-ME-treated cultures led to an increase in osteoblastic activity and a decrease in osteoclast number, respectively. These findings suggest that OPG is not directly involved in 2-ME-mediated anti-proliferative effects in osteosarcoma cells, but rather participates in anti-resorptive functions of 2-ME in bone tumor environment.

Skeletal consequences of deletion of steroid receptor coactivator-2/transcription intermediary factor-2.

Both estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARgamma) regulate bone metabolism, and because steroid receptor coactivator (SRC)-2 (TIF-2) enhances ER and PPARgamma activity, we examined the consequences of deletion of SRC-2 on bone using SRC-2 knock out (KO) mice. Loss of SRC-2 resulted in increased bone mass, with SRC-2 KO mice having 80% higher trabecular bone volume as compared with wild type mice. SRC-2 KO mice also had a marked decrease (by 50%) in bone marrow adipocytes. These data suggested that marrow precursor cells in the SRC-2 KO mice may be resistant to the inhibitory effects of endogenous PPARgamma ligands on bone formation. Consistent with this, compared with cultures from wild type mice, marrow stromal cultures from SRC-2 KO mice formed significantly more mineralized nodules (by 3-fold) in the presence of the PPARgamma agonist, rosiglitazone. Using chromatin immunoprecipitation analysis, we demonstrated that in bone marrow stromal cells, loss of SRC-2 leads to destabilization of the transcription complex at the peroxisome proliferator response elements of a number of PPARgamma target genes, resulting in an overall decrease in the expression of adipocyte-related genes and a marked decrease in adipocyte development. Using ovariectomy with or without estrogen replacement, we also demonstrated that SRC-2 KO mice were partially resistant to the skeletal actions of estrogen. Collectively, these findings indicate that loss of SRC-2 leads to partial skeletal resistance to the ER and PPARgamma, but resistance to PPARgamma is dominant, leading to increased bone mass. Modulating SRC-2 action may, thus, represent a novel therapeutic target for osteoporosis.