Figure 4 - uploaded by Jennifer Tomczak
Content may be subject to copyright.
BRG1 mediates RUNX2 suppression. (A) Representative immunoblot shows increased TRb expression after lentiviral transduction of SW 1736 (SW) cells compared with cells transduced with an empty vector (EV). (B) qRT-PCR results show that transient transfection with a BRG1 expression vector increases BRG1 mRNA levels in both the SW-EV transduced cell line and the SW-TRb transduced cell line. Error bars are SD; significance compared with empty vector control is indicated. (C) qRT-PCR results show changes in RUNX2 mRNA levels after stably transduced cells were transiently transfected with a BRG1 expression vector. Overexpression of BRG1 in the absence of TRb does not repress RUNX2. BRG1 transfection in the presence of TRb represses RUNX2 further than TRb alone. Error bars are SD; significance compared with empty vector control is indicated. *P , 0.05; ***P , 0.001. WT, wild-type.
Source publication
Thyroid hormone receptor beta (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase Brahma Related Gene 1 (BRG1, SMARCA4), a key component of chromatin remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenes...
Contexts in source publication
Context 1
... of three putative prometastatic RUNX2 target genes: AVEGFA, cyclin D1, and MMP9, further emphasizing the importance of maintenance of transcriptional control over RUNX2 for tumor suppression (Fig. 3C). To determine whether reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb ...
Context 2
... the importance of maintenance of transcriptional control over RUNX2 for tumor suppression (Fig. 3C). To determine whether reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb further repressed RUNX2 expression. These results indicate a cooperative mechanism of action ...
Context 3
... reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb further repressed RUNX2 expression. These results indicate a cooperative mechanism of action of TRb and BRG1 for suppression of RUNX2 ...
Context 4
... of three putative prometastatic RUNX2 target genes: AVEGFA, cyclin D1, and MMP9, further emphasizing the importance of maintenance of transcriptional control over RUNX2 for tumor suppression (Fig. 3C). To determine whether reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb ...
Context 5
... the importance of maintenance of transcriptional control over RUNX2 for tumor suppression (Fig. 3C). To determine whether reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb further repressed RUNX2 expression. These results indicate a cooperative mechanism of action ...
Context 6
... reintroduction of BRG1 could rescue RUNX2 repression, we used SW 1736 cells that have been modified to express TRb stably (Fig. 4A). Overexpression of BRG1 by transient transfection in anaplastic thyroid cancer cells (Fig. 4B) did not reduce RUNX2 expression, suggesting that the presence of TRb is necessary for repressive activity of RUNX2 (Fig. 4C). Indeed, expression of TRb alone reduced RUNX2 expression in these cells, whereas reintroduction of both BRG1 and TRb further repressed RUNX2 expression. These results indicate a cooperative mechanism of action of TRb and BRG1 for suppression of RUNX2 ...
Citations
... Thus, we sought to compare the effect of OTUD6A and Brg1 on global gene expression by RNA sequencing (RNA-Seq). As a result, a total of 50 genes, including known Brg1 downstream target genes (SNAI2, IL6, CYP1B1 and RUNX2) [38][39][40][41] , were found to be differentially expressed (34 upregulated and 16 downregulated) by at least 2.0-fold (P < 0.05) upon depletion of either OTUD6A or Brg1 in prostate cancer cells ( Fig. 6i and Supplementary Data 3). However, how these altered genes are involved in Brg1-mediated OTUD6A oncogenic functions needs to be further investigated. ...
Ovarian tumor (OTU) subfamily deubiquitinases are involved in various cellular processes, such as inflammation, ferroptosis and tumorigenesis; however, their pathological roles in prostate cancer (PCa) remain largely unexplored. In this study, we observed that several OTU members displayed genomic amplification in PCa, among which ovarian tumor deubiquitinase 6A (OTUD6A) amplified in the top around 15–20%. Further clinical investigation showed that the OTUD6A protein was highly expressed in prostate tumors, and increased OTUD6A expression correlated with a higher biochemical recurrence risk after prostatectomy. Biologically, wild-type but not a catalytically inactive mutant form of OTUD6A was required for PCa cell progression. In vivo experiments demonstrated that OTUD6A oligonucleotides markedly suppressed prostate tumorigenesis in PtenPC−/− mice and patient-derived xenograft (PDX) models. Mechanistically, the SWI/SNF ATPase subunit Brg1 and the nuclear receptor AR (androgen receptor) were identified as essential substrates for OTUD6A in PCa cells by a mass spectrometry (MS) screening approach. Furthermore, OTUD6A stabilized these two proteins by erasing the K27-linked polyubiquitination of Brg1 and K11-linked polyubiquitination of AR. OTUD6A amplification exhibited strong mutual exclusivity with mutations in the tumor suppressors FBXW7 and SPOP. Collectively, our results indicate the therapeutic potential of targeting OTUD6A as a deubiquitinase of Brg1 and AR for PCa treatment. OTUD6A, a deubiquitinase, is amplified in prostate cancer and correlates with poor survivability, increasing the growth of prostate cancer cell lines and PDX models. OTUD6A stabilizes the expression of Brg1 and AR through the removal of K27- and K11-linked polyubiquination.
... This provided indirect evidence that SWI/SNF chromatin remodeling might be required for target gene repression by TRs. We described an interaction between the SWI/SNF core subunit BRG1 and TR for repression the oncogene RUNX2 (10). Recruitment of the SWI/SNF complex to T 3 -activated promoters has been suggested to be dependent upon interac-2 Nucleic Acids Research, 2022 tions between SRC and p300, rather than a direct interaction between BRG1 and TR (11). ...
Transcriptional regulation in response to thyroid hormone (3,5,3'-triiodo-l-thyronine, T3) is a dynamic and cell-type specific process that maintains cellular homeostasis and identity in all tissues. However, our understanding of the mechanisms of thyroid hormone receptor (TR) actions at the molecular level are actively being refined. We used an integrated genomics approach to profile and characterize the cistrome of TRβ, map changes in chromatin accessibility, and capture the transcriptomic changes in response to T3 in normal human thyroid cells. There are significant shifts in TRβ genomic occupancy in response to T3, which are associated with differential chromatin accessibility, and differential recruitment of SWI/SNF chromatin remodelers. We further demonstrate selective recruitment of BAF and PBAF SWI/SNF complexes to TRβ binding sites, revealing novel differential functions in regulating chromatin accessibility and gene expression. Our findings highlight three distinct modes of TRβ interaction with chromatin and coordination of coregulator activity.
... The final concentration of T 3 in the media was 170 pM. Lentivirally modified SW1736 cells were generated as described (16,23) with either an empty vector (SW-EV) or to overexpress TRβ (SW-TRβ). SW-EV and SW-TRβ were grown in the above conditions with the A c c e p t e d M a n u s c r i p t 6 addition of 2 μg/ml puromycin (Gold Bio, St Louis, MO, USA). ...
Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than six months. Oncogenic alterations in ATC include aberrant PI3K signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and Akt amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRβ) is strongly associated with ATC. TRβ is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85⍺. However, the role of TRβ in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRβ indeed suppresses PI3K signaling in ATC cell lines through unreported genomic mechanisms including a decrease in RTK expression and increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRβ in ATC cell lines enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRβ but shed light into the implication of TRβ status and activation on inhibitor efficacy in ATC tumors.
... Several potential mechanisms of TH-mediated repression have been postulated (Lazar 2003). One model, involving direct TR binding to a so-called negative TRE (nTRE) has been proposed to explain regulation of the Tshb (Wondisford et al. 1989;Carr et al. 1992;Shibusawa et al. 2003) and other genes (Chin et al. 1998;Kim et al. 2005;Nygård et al. 2006;Gillis et al. 2018). A competing model, known as transrepression, suggests that TR binding to nTREs is not via direct DNA binding but, rather, via tethering to another DNAbinding transcription factor such as the AP1 complex (Desbois et al. 1991;Zhang et al. 1991). ...
Thyroid hormones (THs) are powerful regulators of metabolism with major effects on body weight, cholesterol, and liver fat that have been exploited pharmacologically for many years. Activation of gene expression by TH action is canonically ascribed to a hormone-dependent "switch" from corepressor to activator binding to thyroid hormone receptors (TRs), while the mechanism of TH-dependent repression is controversial. To address this, we generated a mouse line in which endogenous TRβ1 was epitope-tagged to allow precise chromatin immunoprecipitation at the low physiological levels of TR and defined high-confidence binding sites where TRs functioned at enhancers regulated in the same direction as the nearest gene in a TRβ-dependent manner. Remarkably, although positive and negative regulation by THs have been ascribed to different mechanisms, TR binding was highly enriched at canonical DR4 motifs irrespective of the transcriptional direction of the enhancer. The canonical NCoR1/HDAC3 corepressor complex was reduced but not completely dismissed by TH and, surprisingly, similar effects were seen at enhancers associated with negatively as well as positively regulated genes. Conversely, coactivator CBP was found at all TH-regulated enhancers, with transcriptional activity correlating with the ratio of CBP to NCoR rather than their presence or absence. These results demonstrate that, in contrast to the canonical "all or none" coregulator switch model, THs regulate gene expression by orchestrating a shift in the relative binding of corepressors and coactivators.
... In addition to the indirect regulation of gene expression, part of the T3 inhibitory effects on Brg1 function seems to occur through some kind of interference between THR and Brg1 chromatin remodeling complex, especially at site A. Although chip analyses are mandatory to confirm this hypothesis, our interpretation is corroborated by previous findings: (1) Brg1 is recruited at nuclear hormone response elements and can directly interact with THR to regulate gene expression either during mammalian development and in the adult life [26][27][28]; (2) epigenetic marks of active chromatin are enriched following T3 treatment in a region embracing site A [29]; (3) in the same region repressive chromatin remodeling marks are enriched by Brg1while epigenetic marks of active chromatin are down-regulated [3]. ...
The LncRNA my-heart (Mhrt) and the chromatin remodeler Brg1 inhibit each other to respectively prevent or favor the maladaptive α-myosin-heavy-chain (Myh6) to β-myosin-heavy-chain (Myh7) switch, so their balance crucially guides the outcome of cardiac remodeling under stress conditions. Even though triiodothyronine (T3) has long been recognized as a critical regulator of the cardiac Myh isoform composition, its role as a modulator of the Mhrt/Brg1 axis is still unexplored. Here the effect of T3 on the Mhrt/Brg1 regulatory circuit has been analyzed in relation with chromatin remodeling and previously identified T3-dependent miRNAs. The expression levels of Mhrt, Brg1 and Myh6/Myh7 have been assessed in rat models of hyperthyroidism or acute myocardial ischemia/reperfusion (IR) treated with T3 replacement therapy. To gain mechanistic insights, in silico analyses and site-directed mutagenesis have been adopted in combination with gene reporter assays and loss or gain of function strategies in cultured cardiomyocytes. Our results indicate a pivotal role of Mhrt over-expression in the T3-dependent regulation of Myh switch. Mechanistically, T3 activates the Mhrt promoter at two putative thyroid hormone responsive elements (TRE) located in a crucial region that is necessary for both Mhrt activation and Brg1-dependent Mhrt repression. This newly identified T3 mode of action requires DNA chromatinization and is critically involved in mitigating the repressive function of the Brg1 protein on Mhrt promoter. In addition, T3 is also able to prevent the Brg1 over-expression observed in the post-IR setting through a pathway that might entail the T3-mediated up-regulation of miR-208a. Taken together, our data evidence a novel T3-responsive network of cross-talking epigenetic factors that dictates the cardiac Myh composition and could be of great translational relevance.
... TRβ has previously been shown to modulate anti-tumorigenic signaling in thyroid cancer in part through repression of driver pathways such as PI3K/AKT (6) and NF-κβ (7); modulation of inflammatory processes (7); and repression of the oncogenes β-Catenin (8,9) and RUNX2 (10,11). These signaling nodes are important contributors to the tumor suppressive activity of TRβ, but they likely do not constitute the entire tumor suppression program. ...
... Anaplastic thyroid cancer cell lines (SW1736, 8505C, OCUT-2, and KTC-2) were cultured in RPMI 1640 growth media with L-glutamine (300 mg/L), sodium pyruvate and nonessential amino acids (1%) (Corning Inc, Corning, NY, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and penicillinstreptomycin (200 IU/L) (Corning) at 37°C, 5% CO 2 , and 100% humidity. Lentivirally modified SW1736 cells were generated as recently described (11) with either an empty vector (SW-EV) or to overexpress TRβ (SW-TRβ). SW-EV and SW-TRβ were grown in the above conditions with the addition of 1 μg/ml puromycin (Gold Bio, St Louis, MO, USA). ...
The thyroid hormone receptor beta (TRβ), a key regulator of cellular growth and differentiation, is frequently dysregulated in cancers. Diminished expression of TRβ is noted in thyroid, breast, and other solid tumors and is correlated with more aggressive disease. Restoration of TRβ levels decreased tumor growth supporting the concept that TRβ could function as a tumor suppressor. Yet, the TRβ tumor suppression transcriptome is not well delineated and the impact of TRβ is unknown in aggressive anaplastic thyroid cancer (ATC). Here, we establish that restoration of TRβ expression in the human ATC cell line SW1736 (SW-TRβ) reduces the aggressive phenotype, decreases cancer stem cell populations and induces cell death in a T3-dependent manner. Transcriptomic analysis of SW-TRβ cells via RNA sequencing revealed distinctive expression patterns induced by ligand-bound TRβ and revealed novel molecular signaling pathways. Of note, liganded TRβ repressed multiple nodes in the PI3K/AKT pathway, induced expression of thyroid differentiation markers, and promoted proapoptotic pathways. Our results further revealed the JAK1–STAT1 pathway as a novel, T3-mediated, antitumorigenic pathway that can be activated in additional ATC lines. These findings elucidate a TRβ-driven tumor suppression transcriptomic signature, highlight unexplored therapeutic options for ATC, and support TRβ activation as a promising therapeutic option in cancers.
Implications
TRβ-T3 induced a less aggressive phenotype and tumor suppression program in anaplastic thyroid cancer cells revealing new potential therapeutic targets.
... Intriguingly, the bone is one of the most common distal sites of metastases in breast cancer [14] and is the second most common site in thyroid metastases [15]. We recently established a novel tumor suppression pathway by which the master regulator of osteoblast development, RUNX2, which functions oncogenically in cancer and promotes invasion and metastasis in thyroid cancer [16,17], is repressed by TRβ in thyroid cells [18,19]. Addition of the ligand, triiodothyronine (T 3 ), enhances the TRβ-mediated transcriptional events, reinforcing this novel pathway. ...
... Transfections were performed using Lipofectamine 3000 (Thermo Scientific) following manufacturer's directions. The pDEST515-FLAG-THRB plasmid is used in the same prior work, as was the negative control vector [18]. We cloned pDEST515-FLAG-THRA using MCF10A cDNA (Supplementary Table 1). ...
Metastatic breast cancer is refractory to conventional therapies and is an end-stage disease. RUNX2 is a transcription factor that becomes oncogenic when aberrantly expressed in multiple tumor types, including breast cancer, supporting tumor progression and metastases. Our previous work demonstrated that the thyroid hormone receptor beta (TRβ) inhibits RUNX2 expression and tumorigenic characteristics in thyroid cells. As TRβ is a tumor suppressor, we investigated the compelling question whether TRβ also regulates RUNX2 in breast cancer. The Cancer Genome Atlas indicates that TRβ expression is decreased in the most aggressive basal-like subtype of breast cancer. We established that modulated levels of TRβ results in corresponding changes in the high levels of RUNX2 expression in metastatic, basal-like breast cells. The MDA-MB-231 triple-negative breast cancer cell line exhibits low expression of TRβ and high levels of RUNX2. Increased expression of TRβ decreased RUNX2 levels. The thyroid hormone-mediated suppression of RUNX2 is TRβ specific as TRα overexpression failed to alter RUNX2 expression. Consistent with these findings, knockdown of TRβ in non-tumor MCF10A mammary epithelial-like cells results in an increase in RUNX2 and RUNX2 target genes. Mechanistically, TRβ directly interacts with the proximal promoter of RUNX2 through a thyroid hormone response element to reduce promoter activity. The TRβ suppression of the oncogene RUNX2 is a signaling pathway shared by thyroid and breast cancers. Our findings provide a novel mechanism for TRβ-mediated tumor suppression in breast cancers. This pathway may be common to many solid tumors and impact treatment for metastatic cancers.
... to chemotherapy.235,238 TRβ, both unliganded and liganded, regulates gene expression via interaction with coregulators and chromatin remodeling complexes.[239][240][241][242][243][244][245] Disruption of TRβ in breast cancer is therefore expected to alter the assembly of cofactorsneeded for transcriptional programming. ...
Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial‐to‐mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer‐compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.
... The pathways that TRb regulates have not yet been thoroughly explored; however, there is evidence that it represses PI3K in both breast and thyroid cancer cells (69,70). TRb also represses transcription of the oncogene RUNX2 in thyroid cancer through an interaction with a chromatin-remodeling complex (71,72). RUNX2 is a driver of both breast and thyroid cancers and common thyroid hormone-mediated mechanisms may be active in both tissues (73,74). ...
Breast and thyroid cancers are two malignancies with highest incidence in women. These cancers often occur metachronously. Women with thyroid cancer are at increased risk for subsequent breast cancer; women with breast cancer have an increased incidence of later development of thyroid cancer, suggesting a common etiology. This bidirectional relationship is reported worldwide; however, the underlying reasons for this co-occurrence are unknown. In this review, we summarize the current epidemiologic evidence and putative mechanisms of these metachronous or synchronous cancers. Key potential causative factors are chemotherapy and radiotherapy of the primary tumor, genetic variants linking the two diseases, hormonal signaling both from the thyroid gland and from estrogens, and lifestyle and environmental factors. There is a critical need for additional epidemiologic studies focused on gender and regional incidence together with molecular investigations on common tumorigenic pathways in these endocrine cancers. Understanding the putative mechanisms will aid in the diagnosis and clinical management of both diseases.
Thyroid hormone (3,5,3’-triiodothyronine, T3) is a key regulator of pituitary gland function. The response to T3 is thought to hinge crucially on interactions of nuclear T3 receptors with enhancers but these sites in pituitary chromatin remain surprisingly obscure. Here, we investigate genome-wide receptor binding in mice using tagged endogenous thyroid hormone receptor β (TRβ) and analyze T3-regulated open chromatin using an anterior pituitary-specific Cre driver (Thrbb2Cre). Strikingly, T3 regulates histone modifications and chromatin opening primarily at sites that maintain TRβ binding regardless of T3 levels rather than at sites where T3 abolishes or induces de novo binding. These sites associate more frequently with T3-activated than T3-suppressed genes. TRβ-deficiency blunts T3-regulated gene expression, indicating that TRβ confers transcriptional sensitivity. We propose a model of gene activation in which poised receptor-enhancer complexes facilitate adjustable responses to T3 fluctuations, suggesting a genomic basis for T3-dependent pituitary function or pituitary dysfunction in thyroid disorders.
