RESUMO
Estrogen signaling is critical for the development and maintenance of healthy bone, and age-related decline in estrogen levels contributes to the development of post-menopausal osteoporosis. Most bones consist of a dense cortical shell and an internal mesh-like network of trabecular bone that respond differently to internal and external cues such as hormonal signaling. To date, no study has assessed the transcriptomic differences that occur specifically in cortical and trabecular bone compartments in response to hormonal changes. To investigate this, we employed a mouse model of post-menopausal osteoporosis (ovariectomy, OVX) and estrogen replacement therapy (ERT). mRNA and miR sequencing revealed distinct transcriptomic profiles between cortical and trabecular bone in the setting of OVX and ERT. Seven miRs were identified as likely contributors to the observed estrogen-mediated mRNA expression changes. Of these, four miRs were prioritized for further study and decreased predicted target gene expression in bone cells, enhanced the expression of osteoblast differentiation markers, and altered the mineralization capacity of primary osteoblasts. As such, candidate miRs and miR mimics may have therapeutic relevance for bone loss resulting from estrogen depletion without the unwanted side effects of hormone replacement therapy and therefore represent novel therapeutic approaches to combat diseases of bone loss.
RESUMO
Menopause is a natural physiological process in older women that is associated with reduced estrogen production and results in increased risk for obesity, diabetes, and osteoporosis. 17α-estradiol (17α-E2) treatment in males, but not females, reverses several metabolic conditions associated with advancing age, highlighting sexually dimorphic actions on age-related pathologies. In this study we sought to determine if 17α-E2 could prevent ovariectomy (OVX)-mediated detriments on adiposity and bone parameters in females. Eight-week-old female C57BL/6J mice were subjected to SHAM or OVX surgery and received dietary 17α-E2 during a six-week intervention period. We observed that 17α-E2 prevented OVX-induced increases in body weight and adiposity. Similarly, uterine weight and luminal cell thickness were decreased by OVX and prevented by 17α-E2 treatment. Interestingly, 17α-E2 prevented OVX-induced declines in tibial metaphysis cancellous bone. And similarly, 17α-E2 improved bone density parameters in both tibia and femur cancellous bone, primarily in OVX mice. In contrast, to the effects on cancellous bone, cortical bone parameters were largely unaffected by OVX or 17α-E2. In the non-weight bearing lumbar vertebrae, OVX reduced trabecular thickness but not spacing, while 17α-E2 increased trabecular thickness and reduced spacing. Despite this, 17α-E2 did improve bone volume/tissue volume in lumbar vertebrae. Overall, we found that 17α-E2 prevented OVX-induced increases in adiposity and changes in bone mass and architecture, with minimal effects in SHAM-operated mice. We also observed that 17α-E2 rescued uterine tissue mass and lining morphology to control levels without inducing hypertrophy, suggesting that 17α-E2 could be considered as an adjunct to traditional hormone replacement therapies.
Assuntos
Densidade Óssea , Estradiol , Idoso , Animais , Estradiol/farmacologia , Estrogênios , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Obesidade , Tamanho do Órgão , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
Cervical cancer (CC) is associated with alterations in immune system balance, which is primarily due to a shift from Th1 to Th2 and the unbalance of Th17/Treg cells. Using in silico DNA copy number analysis, we have demonstrated that ~20% of CC samples exhibit gain of 8q22.3 and 19q13.31; the regions of the genome that encodes the KLF10 and PSG genes, respectively. Gene expression studies demonstrated that there were no alterations in KLF10 mRNA expression, whilst the PSG2 and -5 genes were up-regulated by 1.76 and 3.97-fold respectively in CC compared to normal tissue controls. siRNA and ChIP experiments in SiHa cells have demonstrated that KLF10 participates in immune response through regulation of IL6, IL25 and PSG2 and PSG5 genes. Using cervical tissues from KLF10-/- mice, we have identified down-regulation of PSG17, -21 and -23 and IL11. These results suggest that KLF10 may regulate immune system response genes in cervical cancer among other functions. KLF10 and PSG copy number variations and alterations in mRNA expression levels could represent novel molecular markers in CC.
Assuntos
Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Glicoproteínas beta 1 Específicas da Gravidez/genética , Neoplasias do Colo do Útero/genética , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Feminino , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Glicoproteínas beta 1 Específicas da Gravidez/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/imunologiaRESUMO
TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone.
Assuntos
Proteínas de Ligação a DNA/deficiência , Estrogênios/farmacologia , Glicoproteínas/metabolismo , Osteócitos/efeitos dos fármacos , Esqueleto/metabolismo , Fatores de Transcrição/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Animais , Densidade Óssea/efeitos dos fármacos , Densidade Óssea/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/metabolismo , Feminino , Marcadores Genéticos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos Knockout , Osteócitos/metabolismo , Ovariectomia/métodos , Esqueleto/efeitos dos fármacosRESUMO
Endoxifen, the primary active metabolite of tamoxifen, is currently being investigated as a novel endocrine therapy for the treatment of breast cancer. Tamoxifen is a selective estrogen receptor modulator that elicits potent anti-breast cancer effects. However, long-term use of tamoxifen also induces bone loss in premenopausal women and is associated with an increased risk of endometrial cancer in postmenopausal women. For these reasons, we have used a rat model system to comprehensively characterize the impact of endoxifen on the skeleton and uterus. Our results demonstrate that endoxifen elicits beneficial effects on bone in ovary-intact rats and protects against bone loss following ovariectomy. Endoxifen is also shown to reduce bone turnover in both ovary-intact and ovariectomized rats at the cellular and biochemical levels. With regard to the uterus, endoxifen decreased uterine weight but maintained luminal epithelial cell height in ovariectomized animals. Within luminal epithelial cells, endoxifen resulted in differential effects on the expression levels of estrogen receptors α and ß as well as multiple other genes previously implicated in regulating epithelial cell proliferation and hypertrophy. These studies analyze the impact of extended endoxifen exposure on both bone and uterus using a Food and Drug Administration-recommended animal model. Although endoxifen is a more potent breast cancer agent than tamoxifen, the results of the present study demonstrate that endoxifen does not induce bone loss in ovary-intact rats and that it elicits partial agonistic effects on the uterus and skeleton in ovariectomized animals.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Útero/efeitos dos fármacos , Animais , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Endométrio/patologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/metabolismo , Feminino , Tamanho do Órgão , Osteoporose/induzido quimicamente , Ovariectomia , Ratos , Moduladores Seletivos de Receptor Estrogênico/efeitos adversos , Tamoxifeno/efeitos adversos , Tamoxifeno/farmacologia , Útero/metabolismo , Útero/patologiaRESUMO
TGFß-SMAD signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. Longstanding challenges in resolving this functional dichotomy may uncover new strategies to treat advanced carcinomas. The Krüppel-like transcription factor, KLF10, is a pivotal effector of TGFß/SMAD signaling that mediates antiproliferative effects of TGFß. In this study, we show how KLF10 opposes the prometastatic effects of TGFß by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). KLF10 depletion accentuated induction of EMT as assessed by multiple metrics. KLF10 occupied GC-rich sequences in the promoter region of the EMT-promoting transcription factor SLUG/SNAI2, repressing its transcription by recruiting HDAC1 and licensing the removal of activating histone acetylation marks. In clinical specimens of lung adenocarcinoma, low KLF10 expression associated with decreased patient survival, consistent with a pivotal role for KLF10 in distinguishing the antiproliferative versus prometastatic functions of TGFß. Our results establish that KLF10 functions to suppress TGFß-induced EMT, establishing a molecular basis for the dichotomy of TGFß function during tumor progression. Cancer Res; 77(9); 2387-400. ©2017 AACR.
Assuntos
Adenocarcinoma/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Transição Epitelial-Mesenquimal/genética , Retroalimentação Fisiológica , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , Fator de Crescimento Transformador beta/genética , Células A549 , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Humanos , Neoplasias Pulmonares/patologia , Camundongos Knockout , Pacientes , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição da Família Snail/genéticaRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Antagonistas de Estrogênios/farmacologia , Receptor beta de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/genética , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Osso e Ossos/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Tamoxifeno/análogos & derivados , Animais , Antineoplásicos Hormonais/farmacologia , Sequência de Bases , Primers do DNA , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Reação em Cadeia da Polimerase , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
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.
Assuntos
Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Estrogênios/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais , Fatores de Transcrição/biossíntese , Animais , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , Osso e Ossos/patologia , Proteínas de Ligação a DNA/genética , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Antagonistas de Estrogênios/metabolismo , Tamoxifeno/análogos & derivados , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Análise por Conglomerados , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligação Proteica , Reprodutibilidade dos Testes , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.