Gillian H. Little

Nuclear Calcium/Calmodulin-dependent Protein Kinase IIδ Preferentially Transmits Signals to Histone Deacetylase 4 in Cardiac Cells

Class II histone deacetylases (HDACs) act as repressors of cardiac hypertrophy, an adaptative response of the heart characterized by a reprogramming of fetal cardiac genes. Prolonged hypertrophy often leads to dilated cardiomyopathy and heart failure. Upstream endogenous regulators of class II HDACs that regulate hypertrophic growth are just beginning to emerge. HerewedemonstratethattheδBisoformofcalcium/calmodulin-dependent protein kinase II (CaMKIIδB), known to promote cardiac hypertrophy, transmits signals specifically to HDAC4 but not other class II HDACs. CaMKIIδB efficiently phosphorylates both a glutathione S-transferase (GST)-HDAC4 fragment spanning amino acids 207–311 and full-length FLAG-HDAC4 but not the equivalents in HDAC5. Although previous studies in skeletal muscle cells have shown that HDAC4 lacking serine 246 cannot be phosphorylated by CaMKI/IV, a similar mutant is still phosphorylated by CaMKIIδB. Importantly, mutation of serine 210 to alanine totally abolishes phosphorylation of the GST fragment and significantly reduces phosphorylation of full-length HDAC by CaMKIIδB. RNA interference knockdown of CaMKIIδB prevents the effects of hypertrophic stimuli. Overexpression of CaMKIIδB in primary neonatal cardiomyocytes increases the activity of the Mef2 transcription factor and completely rescues HDAC4-mediated repression of MEF2 but only partially rescues inhibition by HDAC5 or the HDAC4 S210A mutant. CaMKIIδB strongly interacts with HDAC4 in cells but not with HDAC5. These results demonstrate that CaMKIIδB preferentially targets HDAC4, and this involves serine 210. These findings identify HDAC4 as a specific downstream substrate of CaMKIIδB in cardiac cells and have broad applications for the signaling pathways leading to cardiac hypertrophy and heart failure.

Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2

IntroductionIn contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.MethodsMCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.ResultsRunx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.ConclusionsE2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level

Runx2 Controls a Feed-forward loop between Androgen and Prolactin-induced Protein (PIP) in Stimulating T47D Cell Proliferation

Prolactin‐Induced Protein (PIP) is a small polypeptide expressed by breast and prostate cancer (BCa, PCa) cells. However, both the regulation of PIP expression and its function in cancer cells are poorly understood. Using BCa and PCa cells, we found that Runx2, a pro‐metastatic transcription factor, functionally interacts with the Androgen Receptor (AR) to regulate PIP expression. Runx2 expression in C4‐2B PCa cells synergized with AR to promote PIP expression, whereas its knockdown in T47D BCa cells abrogated basal as well as hormone stimulated PIP expression. Chromatin immunoprecipitation (ChIP) assays showed that Runx2 and AR co‐occupied an enhancer element located ∼11 kb upstream of the PIP open reading frame, and that Runx2 facilitated AR recruitment to the enhancer. PIP knockdown in T47D cells compromised DHT‐stimulated expression of multiple AR target genes including PSA, FKBP5, FASN, and SGK1. The inhibition of AR activity due to loss of PIP was attributable at least in part to abrogation of its nuclear translocation. PIP knockdown also suppressed T47D cell proliferation driven by either serum growth factors or dihydrotestosterone (DHT). Our data suggest that Runx2 controls a positive feedback loop between androgen signaling and PIP, and pharmacological inhibition of PIP may be useful to treat PIP positive tumors. J. Cell. Physiol. 227: 2276–2282, 2012.

Critical Role of Nuclear Calcium/Calmodulin-dependent Protein Kinase IIδB in Cardiomyocyte Survival in Cardiomyopathy

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in cardiac contractility and heart disease. However, the specific role of alternatively spliced variants of CaMKII in cardiac disease and apoptosis remains poorly explored. Here we report that the δB subunit of CaMKII (CaMKIIδB), which is the predominant nuclear isoform of calcium/calmodulin-dependent protein kinases in heart muscle, acts as an anti-apoptotic factor and is a novel target of the antineoplastic and cardiomyopathic drug doxorubicin (Dox (adriamycin)). Hearts of rats that develop cardiomyopathy following chronic treatment with Dox also show down-regulation of CaMKIIδB mRNA, which correlates with decreased cardiac function in vivo, reduced expression of sarcomeric proteins, and increased tissue damage associated with Dox cardiotoxicity. Overexpression of CaMKIIδB in primary cardiac cells inhibits Dox-mediated apoptosis and prevents the loss of the anti-apoptotic protein Bcl-2. Specific silencing of CaMKIIδB by small interfering RNA prevents the formation of organized sarcomeres and decreases the expression of Bcl-2, which all mimic the effect of Dox. CaMKIIδB is required for GATA-4-mediated co-activation and binding to the Bcl-2 promoter. These results reveal that CaMKIIδB plays an essential role in cardiomyocyte survival and provide a mechanism for the protective role of CaMKIIδB. These results suggest that selective targeting of CaMKII in the nuclear compartment might represent a strategy to regulate cardiac apoptosis and to reduce Dox-mediated cardiotoxicity.

Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial–mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS–PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2s transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion.

Recruitment of coregulator G9a by Runx2 for selective enhancement or suppression of transcription.

Runx2, best known for its role in regulating osteoblast‐specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4‐2B/Rx2dox prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus‐dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co‐immunoprecipitation, GST‐pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer‐related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity. J. Cell. Biochem. 113: 2406–2414, 2012.

Differential effects of RUNX2 on the androgen receptor in prostate cancer: synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness

Changes to androgen signaling during prostate carcinogenesis are associated with both inhibition of cellular differentiation and promotion of malignant phenotypes. The androgen receptor (AR)-binding transcription factor RUNX2 has been linked to prostate cancer progression but the underlying mechanisms have not been fully defined. In this study, we investigated the genome-wide influence of RUNX2 on androgen-induced gene expression and AR DNA binding in prostate cancer cells. RUNX2 inhibited the androgen response partly by promoting the dissociation of AR from its target genes such as the tumor suppressor NKX3-1. However, AR activity persists in the presence of RUNX2 at other AR target genes, some of which are cooperatively stimulated by androgen and RUNX2 signaling. These genes are associated with putative enhancers co-occupied by AR and RUNX2. One such gene, the invasion-promoting Snail family transcription factor SNAI2, was co-activated by AR and RUNX2. Indeed, these two transcription factors together, but neither alone stimulated prostate cancer cell invasiveness, which could be abolished by SNAI2 silencing. Furthermore, an immunohistochemical analysis of SNAI2 in archived primary prostate cancer specimens revealed a correlation with the RUNX2 histoscore, and simultaneous strong staining for SNAI2, RUNX2, and AR (but not any pair alone) was associated with disease recurrence. Overall, our findings suggest cooperation between AR and RUNX in the stimulation of oncogenes such as SNAI2, which might be targeted for individualized prostate cancer therapy.

RUNX1 prevents oestrogen-mediated AXIN1 suppression and β-catenin activation in ER-positive breast cancer

Recent high-throughput studies revealed recurrent RUNX1 mutations in breast cancer, specifically in oestrogen receptor-positive (ER+) tumours. However, mechanisms underlying the implied RUNX1-mediated tumour suppression remain elusive. Here, by depleting mammary epithelial cells of RUNX1 in vivo and in vitro, we demonstrate combinatorial regulation of AXIN1 by RUNX1 and oestrogen. RUNX1 and ER occupy adjacent elements in AXIN1s second intron, and RUNX1 antagonizes oestrogen-mediated AXIN1 suppression. Accordingly, RNA-seq and immunohistochemical analyses demonstrate an ER-dependent correlation between RUNX1 and AXIN1 in tumour biopsies. RUNX1 loss in ER+ mammary epithelial cells increases β-catenin, deregulates mitosis and stimulates cell proliferation and expression of stem cell markers. However, it does not stimulate LEF/TCF, c-Myc or CCND1, and it does not accelerate G1/S cell cycle phase transition. Finally, RUNX1 loss-mediated deregulation of β-catenin and mitosis is ameliorated by AXIN1 stabilization in vitro, highlighting AXIN1 as a potential target for the management of ER+ breast cancer.

Initial Characterization of Osteoblast Differentiation and Loss of RUNX2 Stability in the Newly Established SK11 Human Embryonic Stem Cell‐Derived Cell Line

We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cells display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include osterix, α1(I) collagen, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. Similar to well‐characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)‐inducible FLAG‐tagged RUNX2 into SK11 cells. Dox‐mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co‐treatment with BMP‐2. Like the endogenous RUNX2, expression of the virally coded FLAG‐RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day‐5 versus day‐3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here by the regulation of matrix Gla Protein, may have significant biomedical implications. J. Cell. Physiol. 230: 237–241, 2015.

High-throughput screen for inhibitors of androgen receptor-RUNX2 transcriptional regulation in prostate cancer

Runt-related transcription factor 2 (RUNX2) plays a critical role in prostate cancer progression. RUNX2 interacts with the androgen receptor (AR) and modulates its transcriptional activity in a locus-specific manner. RUNX2 and AR synergistically stimulate a subset of genes, including the pro-oncogene snail family zinc finger 2 (SNAI2). AR-RUNX2 signaling cooperatively induces invasiveness of prostate cancer cells via SNAI2; and coexpression of AR, RUNX2, and SNAI2 in prostate cancer biopsy samples predicts disease recurrence. Competitive inhibition of AR alone could not disrupt the synergistic activation of SNAI2. We therefore established a phenotypic cell-based screening assay for compounds that could inhibit AR-RUNX2 synergistic activity either directly or indirectly. This assay was used to screen 880 compounds as a proof of concept, resulting in identification of several compounds that disrupted the synergistic stimulation of genes. Further investigation suggested the involvement of epidermal growth factor receptor (EGFR) signaling in AR/RUNX2 synergistic activity. Our assay is amenable to high-throughput screening and can be used to identify inhibitors of the AR-RUNX2 interaction in prostate cancer cells.