Kevin S. Pitel

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency.

Expression of p16Ink4a and p19Arf 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 p16Ink4a or p19Arf 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 p16Ink4a and p19Arf. Inactivation of p16Ink4a in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19Arf 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 p16Ink4a is an effector and p19Arf an attenuator of senescence and ageing in these tissues.

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.

ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer

BackgroundThe role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 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.MethodsNuclear and cytoplasmic expression patterns of ERβ1 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 ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.ResultsNuclear ERβ1 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, ERβ1 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, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.ConclusionsUsing a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.

TGFβ Inducible Early Gene-1 Plays an Important Role in Mediating Estrogen Signaling in the Skeleton

TGFβ 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.

Krüppel-like Transcription Factor KLF10 Suppresses TGFβ-Induced Epithelial-to-Mesenchymal Transition via a Negative Feedback Mechanism

TGFβ-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 TGFβ/SMAD signaling that mediates antiproliferative effects of TGFβ. In this study, we show how KLF10 opposes the prometastatic effects of TGFβ 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 TGFβ. Our results establish that KLF10 functions to suppress TGFβ-induced EMT, establishing a molecular basis for the dichotomy of TGFβ function during tumor progression. Cancer Res; 77(9); 2387-400. ©2017 AACR.

TIEG1 enhances Osterix expression and mediates its induction by TGFβ and BMP2 in osteoblasts.

Deletion of TIEG1/KLF10 in mice results in an osteopenic skeletal phenotype with significant decreases in both bone mineral density and content throughout the skeleton. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display numerous changes in gene expression and exhibit significant delays in their mineralization rates relative to wild-type (WT) controls. Here, we demonstrate that loss of TIEG1 expression in osteoblasts results in decreased levels of Osterix mRNA. Suppression of TIEG1 expression in WT osteoblasts leads to decreased Osterix expression while restoration of TIEG1 expression in TIEG1 KO osteoblasts results in increased levels of Osterix. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Osterix promoter and demonstrate that the zinc finger-containing DNA binding domain of TIEG1 is necessary for this regulation. Furthermore, we reveal that TIEG1 expression is essential for the induction of Osterix expression by important bone-related cytokines such as TGFβ and BMP2 in osteoblast cells. Taken together, these data implicate an important role for TIEG1 in regulating the expression of Osterix, a master regulator of osteoblast differentiation and bone formation, and suggest that decreased expression of Osterix, as well as impaired TGFβ and BMP2 signaling, contribute to the observed osteopenic bone phenotype of TIEG1 KO mice.

TIEG1 modulates β-catenin sub-cellular localization and enhances Wnt signaling in bone

Abstract We have previously demonstrated that TGFβ Inducible Early Gene-1 (TIEG1), also known as KLF10, plays important roles in mediating skeletal development and homeostasis in mice. TIEG1 has also been identified in clinical studies as one of a handful of genes whose altered expression levels or allelic variations are associated with decreased bone mass and osteoporosis in humans. Here, we provide evidence for the first time that TIEG1 is involved in regulating the canonical Wnt signaling pathway in bone through multiple mechanisms of action. Decreased Wnt signaling in the absence of TIEG1 expression is shown to be in part due to impaired β-catenin nuclear localization resulting from alterations in the activity of AKT and GSK-3β. We also provide evidence that TIEG1 interacts with, and serves as a transcriptional co-activator for, Lef1 and β-catenin. Changes in Wnt signaling in the setting of altered TIEG1 expression and/or activity may in part explain the observed osteopenic phenotype of TIEG1 KO mice as well as the known links between TIEG1 expression levels/allelic variations and patients with osteoporosis.

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.

Erratum: Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency (Nature Cell Biology (2008) 10 (825-836))

In the version of this Article originally published, in the legend for Fig. 4, the statement “Data in a and d represent the mean of two independent experiments” should have read: “Data in a and e represent the mean of two independent experiments”. This error has been corrected in both the HTML and PDF versions of the Article. Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming Gaoyang Liang, Jin He and Yi Zhang

TIEG and estrogen modulate SOST expression in the murine skeleton

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.