Citations

This report describes the relationship between bone formation and mRNA levels for selected bone proteins. Dynamic bone histomorphometry was used to measure bone formation in tibial periosteum of male rats from weanling (3 wk) to 52 wk old. Northern blot analysis of freshly isolated periosteal cells from the long bones was used to determine steady-state mRNA levels for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAP), the bone matrix proteins osteocalcin (BGP), and prepro-alpha-2 (I) chain of type 1 precollagen (collagen), the osteoblast marker enzyme alkaline phosphatase (AP), and the osteoblast-derived signaling factor (growth factor) transforming growth factor-beta (TGF-beta). Radial growth at the tibial diaphysis achieved a maximum value in 8-wk-old rats and decreased progressively with age thereafter. This age-related decrease in the radial growth rate was initially due to reduced osteoblast activity; however, in older rats (greater than 17 wk old) reduced osteoblast number contributed to the decrease in bone formation. There was a strong correlation between the steady-state mRNA level for collagen and the periosteal bone formation rate. In contrast, the mRNA levels for the other bone proteins were more weakly correlated (TGF-beta and AP) or not correlated (BGP). These results suggest that the decreased bone matrix synthesis by periosteal cells in long bones of maturing rats is due to decreased expression of genes for bone matrix proteins.

Abstract The Transforming Growth Factor-β (TGFβ)/SMAD signaling pathway can function as either a tumor suppressor or metastasis promoter during tumor progression. In normal epithelial cells and early stages of epithelial tumorigenesis TGFβ functions as a tumor suppressor to decrease cell proliferation or induce apoptosis. However, during malignant progression tumor cells no longer respond to the anti-proliferative effects of TGFβ, but instead undergo an epithelial-to-mesenchymal transition (EMT) whereby cells acquire a migratory and invasive phenotype which promotes tumor metastasis. Resolution of the dichotomy in TGFβ function and a further understanding of its tumor suppressor and metastasis promoting functions may uncover new strategies for the treatment of epithelial cancers. In previous studies we demonstrated an important role of the TGFβ-Inducible Early Gene-1 (TIEG1)/Krüppel-like Factor-10 (KLF10) as a central regulator of TGFβ/SMAD signaling and the anti-proliferative functions of TGFβ. In this study we examined the role of KLF10 in controlling the TGFβ-induced EMT in human lung adenocarcinoma cells. We show that depletion of KLF10 results in a more pronounced induction of EMT in human A549 lung adenocarcinoma cells as assessed by quantitative real-time PCR, changes in cellular morphology and immunofluorescence microscopy, and transcriptome-wide (RNA-seq) analyses. Moreover, chromatin immunoprecipitation (ChIP) and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis shows that KLF10 directly binds to GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2 to repress its transcription. Consistent with these findings, an analysis of KLF10 and SNAI2 expression in lung cancer revealed that while KLF10 levels are decreased in lung cancer vs. normal samples, SNAI2 levels are increased. Additional ChIP studies showed that KLF10 recruits HDAC1 to the SNAI2 promoter and is required for the removal of activating histone acetylation marks. These findings reveal a previously unknown function of KLF10 in suppressing TGFβ-induced EMT and represent a significant advancement in the understanding the molecular dichotomy of TGFβ function during tumor progression. Citation Format: Vivek Kumar Mishra, Vijayalakshmi Kari, Malayannan Subramaniam, Simon J. Baumgart, Sankari Nagarajan, Florian Wegwitz, Thomas C. Spelsberg, John R. Hawse, Steven A. Johnsen. Krüppel-like Transcription Factor-10 (KLF10) suppresses the TGFβ-induced epithelial-to-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A03.

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1 × 10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1×10(-9)-1×10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.

Abstract Background: While the biological functions and clinical importance of ERα are well understood in breast cancer, much less is known about its most closely related family member, ERβ, particularly in the setting of triple negative disease. Additionally, the ability to therapeutically target ERβ in triple negative breast cancer (TNBC) has not been fully explored. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in a cohort of 71 TNBCs. To further define the biological functions of ERβ in TNBC, novel ERβ expressing triple negative cell lines (MDA-MB-231 and Hs578T) were developed and comprehensively characterized at the level of global gene expression profiling, modulation of important biological pathways, cellular proliferation and response to targeted therapies. Results: In TNBCs from 71 patients, nuclear and cytoplasmic ERβ was detected at moderate to high levels in 24% and 32% of cases respectively. This moderate to high expression of both nuclear and cytoplasmic ERβ was associated with higher levels of Ki67. Of the 17 tumors expressing ERβ, 13 (76%) were negative for androgen receptor expression. In the triple negative MDA-MB-231-ERβ and Hs578T-ERβ cell lines, expression of ERβ led to inhibition of proliferation in response to both estrogen and multiple ERβ specific agonists. Microarray analysis and RT-PCR profiling of these cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors, while suppressing the expression of many interleukins. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells inhibited the proliferation rates and blocked TGFβ signaling in non-ERβ expressing TNBC cells, effects that were completely reversed following depletion of cystatins from the conditioned media. Conclusions: ERβ is expressed in a substantial proportion of TNBCs, and most do not express the androgen receptor. In TNBC, where targeted therapies are lacking, our data suggest that estrogen or ERβ specific agonists would be expected to elicit anti-tumor effects when ERβ is expressed. These tumor suppressive effects of ERβ appear to be mediated in part through the actions of cystatins, and their inhibition of TGFβ signaling, suggesting that this family of secreted proteins may represent novel biomarkers for monitoring ERβ specific drug responsiveness and/or patient outcomes. Citation Format: Jordan M Reese, Malayannan Subramaniam, Vera J Suman, Xianglin Wu, Vivian Negron, Anne Gingery, Kevin S Pitel, Sejal S Shah, Heather E Cunliffe, Ann E McCullough, Barbara A Pockaj, Thomas C Spelsberg, Matthew P Goetz, James N Ingle, John R Hawse. Therapeutic targeting of ERβ in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-06.

Abstract Background: Estrogen receptor beta (ERβ), unlike ERα, classically functions as a tumor suppressor in vitro. However, ERβ's biological functions in vivo and predictive/prognostic value in breast cancer are controversial. Methods: Expression of ERβ protein was determined using a well characterized and validated ERβ specific monoclonal antibody that only recognizes the full-length form of this receptor (PPG5/10) in the following 3 cohorts: 1) a cohort with all breast cancer subtypes (n = 182), 2) a prospective NCCTG adjuvant tamoxifen trial for postmenopausal women with ERα positive breast cancer with long-term follow-up (n = 198) and 3) a cohort of 80 triple negative breast cancers (TNBCs). To elucidate the biological functions of ERβ in breast cancer, novel ERβ expressing MCF7 and MDA-MB-231 cell lines were developed and characterized using multiple techniques and were examined for responsiveness to various ERβ targeted therapies. Results: About one-third of all breast tumors, regardless of sub-type, were shown to express nuclear ERβ and this expression was independent of ERα or HER2. In the NCCTG 89-30-52 cohort, breast cancer recurrence rates were significantly associated with ERβ protein expression with 10 year recurrence rates of 25%, 15% and 4% for zero, low or high levels of ERβ expression respectively. Interestingly, in TNBCs, nuclear ERβ was expressed at intermediate or high levels in 24% of tumors. In the triple negative MDA-MB-231 cell line, expression of ERβ led to inhibition of proliferation and induction of apoptosis in response to estrogen and multiple ERβ specific agonists. Conversely, these same treatments induced proliferation of ERβ-expressing MCF7 cells which endogenously express ERα. However, ERβ expression sensitized MCF7 cells to the anti-proliferative effects of anti-estrogens. Microarray analysis and RT-PCR profiling of MDA-MB-231-ERβ cells revealed that estrogen and ERβ agonists highly induced the expression of multiple cystatins, a family of small secreted cysteine protease inhibitors which function as tumor suppressors, and potently inhibited canonical TGFβ signaling. Conditioned media isolated from estrogen or ERβ agonist treated MDA-MB-231-ERβ cells suppressed the proliferation rates and inhibited TGFβ signaling in other TNBC cell lines, effects that were completely reversed following the depletion of cystatins from the conditioned media. Conclusions: These data demonstrate that ERβ is expressed in a substantial proportion of breast cancers and may have value as a predictive and/or prognostic biomarker. Therapeutic targeting of ERβ may have clinical benefit in multiple breast cancer sub-types; however, the specific drug of choice may vary based on ERα status. Specifically, we have demonstrated that ERβ expression in ERα+ MCF7 cells sensitizes them to the effectiveness of anti-estrogens, an observation that was confirmed in women enrolled in a prospective adjuvant tamoxifen trial. In TNBCs, where targeted therapies are lacking, our data suggest that targeting ERβ with either estrogen or ERβ specific agonists will elicit anti-tumor effects through the induction of cystatins and inhibition of TGFβ signaling resulting in tumor regression and improved patient outcomes. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-03.

Abstract Background: Commonly used endocrine therapies for breast cancer, such as aromatase inhibitors in postmenopausal women and tamoxifen in premenopausal women, have deleterious effects on bone mineral density. Therefore, the identification of novel cancer therapies which either maintain or improve bone mass are of clinical need. We and others have previously demonstrated that endoxifen is the most active tamoxifen metabolite responsible for eliciting the anti-cancer effects of this drug and that endoxifen concentrations are an important factor with regard to tamoxifen efficacy. These studies have led to the development of endoxifen as a novel anti-breast cancer drug for which phase I clinical trials are now underway. At present, there are no data regarding endoxifen's effects on bone. Methods: The effects of endoxifen on osteoblast gene expression profiles were compared to that of estrogen, tamoxifen, raloxifene and lasofoxifene by microarray and RT-PCR analyses in both estrogen receptor alpha (ERα) and ERβ expressing cell lines. The in vivo effects of an anti-cancer dose of endoxifen (50mg/kg/day) on the skeleton were first analyzed in 3-month-old ovariectomized C57BL/6 mice using Dual-energy X-ray absorptiometry, peripheral Quantitative Computed Tomography, micro-Computed Tomography and histomorphometry. In a second set of studies, a pre-clinical rat model was used to determine the effects of endoxifen (10mg/kg/day) on the skeleton in both a pre- and post-menopausal setting. Results: Endoxifen treatment of ERα and ERβ expressing mouse osteoblast cells led to dramatically different gene expression profiles when compared to that of estrogen and other anti-estrogens. In ovariectomized mice, daily administration of endoxifen led to significant increases in bone mineral density and content throughout the skeleton relative to vehicle control treated animals. The numbers and activity of both osteoblasts and osteoclasts were also found to be significantly higher in endoxifen treated mice. In the pre-clinical model system, endoxifen treatment of 4 month-old ovariectomized Sprague-Dawley rats significantly protected against bone loss following estrogen depletion primarily due to suppression of osteoclast mediated bone resorption. Importantly, in sham operated rats (thus retaining ovarian function), endoxifen treatment enhanced bone volume and trabecular thickness and did not suppress osteoclast activity. Conclusions: These data are the first to examine the effects of the novel breast cancer therapy, endoxifen, on bone and reveal that the molecular mechanisms of action of this compound are substantially different than that of other SERMs. Endoxifen was shown to protect against bone loss following estrogen depletion in both mice and rats and interestingly, enhanced bone mass in ovary intact rats, an observation that is in stark contrast to the known effects of tamoxifen which induces bone loss in the “pre-menopausal” setting. These studies suggest that endoxifen may have superior bone-beneficial effects compared to tamoxifen, and if efficacy is confirmed in later phase trials, endoxifen may represent a better drug of choice for a sub-set of breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-01.