Runx1 Deficiency Protects Against Adverse Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown. METHODS: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1-deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels. RESULTS: Runx1-deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca2+-ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca2+-ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum-mediated calcium release, preserving cardiomyocyte contraction after MI. CONCLUSIONS: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI.

Runx Genes in Breast Cancer and the Mammary Lineage.

A full understanding of RUNX gene function in different epithelial lineages has been thwarted by the lethal phenotypes observed when constitutively knocking out these mammalian genes. However temporal expression of the Runx genes throughout the different phases of mammary gland development is indicative of a functional role in this tissue. A few studies have emerged describing how these genes impact on the fate of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential, with implications for the transformed state. The importance of the RUNX/CBFß core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFß appearing in a comprehensive gene list of predicted breast cancer driver mutations. Nonetheless, the evidence to date shows that the RUNX genes can have dualistic outputs with respect to promoting or constraining breast cancer phenotypes, and that this may be aligned to individual subtypes of the clinical disease. We take this opportunity to review the current literature on RUNX and CBFß in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

The RUNX genes: gain or loss of function in cancer.

The RUNX genes have come to prominence recently because of their roles as essential regulators of cell fate in development and their paradoxical effects in cancer, in which they can function either as tumour-suppressor genes or dominant oncogenes according to context. How can this family of transcription factors have such an ambiguous role in cancer? How and where do these genes impinge on the pathways that regulate growth control and differentiation? And what is the evidence for a wider role for the RUNX genes in non-haematopoietic cancers?

RUNX2 in mammary gland development and breast cancer.

Runx2 is best known as an essential factor in osteoblast differentiation and bone development but, like many other transcription factors involved in development, is known to operate over a much wider tissue range. Our understanding of these other aspects of Runx2 function is still at a relatively early stage and the importance of its role in cell fate decisions and lineage maintenance in non-osseous tissues is only beginning to emerge. One such tissue is the mammary gland, where Runx2 is known to be expressed and participate in the regulation of mammary specific genes. Furthermore, differential and temporal expression of this gene is observed during mammary epithelial differentiation in vivo, strongly indicative of an important functional role. Although the precise nature of that role remains elusive, preliminary evidence hints at possible involvement in the regulation of mammary stem and/or progenitor cells. As with many genes important in regulating cell fate, RUNX2 has also been linked to metastatic cancer where in some established breast cell lines, retention of expression is associated with a more invasive phenotype. More recently, expression analysis has been extended to primary breast cancers where high levels of RUNX2 align with a specific subtype of the disease. That RUNX2 expression correlates with the so called "Triple Negative" subtype is particularly interesting given the known cross talk between Runx2 and estrogen receptor signaling pathways. This review summaries our current understanding of Runx2 in mammary gland development and cancer, and postulates a role that may link both these processes.

The RUNX/CBFß Complex in Breast Cancer: A Conundrum of Context.

Dissecting and identifying the major actors and pathways in the genesis, progression and aggressive advancement of breast cancer is challenging, in part because neoplasms arising in this tissue represent distinct diseases and in part because the tumors themselves evolve. This review attempts to illustrate the complexity of this mutational landscape as it pertains to the RUNX genes and their transcription co-factor CBFß. Large-scale genomic studies that characterize genetic alterations across a disease subtype are a useful starting point and as such have identified recurring alterations in CBFB and in the RUNX genes (particularly RUNX1). Intriguingly, the functional output of these mutations is often context dependent with regards to the estrogen receptor (ER) status of the breast cancer. Therefore, such studies need to be integrated with an in-depth understanding of both the normal and corrupted function in mammary cells to begin to tease out how loss or gain of function can alter the cell phenotype and contribute to disease progression. We review how alterations to RUNX/CBFß function contextually ascribe to breast cancer subtypes and discuss how the in vitro analyses and mouse model systems have contributed to our current understanding of these proteins in the pathogenesis of this complex set of diseases.

Ribonucleicacid interference or small molecule inhibition of Runx1 in the border zone prevents cardiac contractile dysfunction following myocardial infarction.

AIMS: Myocardial infarction (MI) is a major cause of death worldwide. Effective treatments are required to improve recovery of cardiac function following MI, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile region bordering an infarct is functionally distinct from the remote surviving myocardium and is a determinant of adverse remodelling and cardiac contractility. Expression of the transcription factor RUNX1 is increased in the border zone 1-day after MI, suggesting potential for targeted therapeutic intervention. OBJECTIVE: This study sought to investigate whether an increase in RUNX1 in the border zone can be therapeutically targeted to preserve contractility following MI. METHODS AND RESULTS: In this work we demonstrate that Runx1 drives reductions in cardiomyocyte contractility, calcium handling, mitochondrial density, and expression of genes important for oxidative phosphorylation. Both tamoxifen-inducible Runx1-deficient and essential co-factor common ß subunit (Cbfß)-deficient cardiomyocyte-specific mouse models demonstrated that antagonizing RUNX1 function preserves the expression of genes important for oxidative phosphorylation following MI. Antagonizing RUNX1 expression via short-hairpin RNA interference preserved contractile function following MI. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with CBFß. CONCLUSIONS: Our results confirm the translational potential of RUNX1 as a novel therapeutic target in MI, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

Retroviral insertion sites and cancer: fountain of all knowledge?

Retroviral gene tagging is enjoying a renaissance as a gene discovery method since the completion of the draft mouse genome sequence. The potential of this approach to elucidate the genetic basis of cancer is reviewed in the light of a series of recent papers that report the results of high-throughput screens.

Runx2 in normal tissues and cancer cells: A developing story.

The Runx transcription factors are essential for mammalian development, most notably in the haematopoietic and osteogenic lineages. Runx1 and its binding partner, CBFbeta, are frequently targeted in acute leukaemia but evidence is accumulating that all three Runx genes may have a role to play in a wider range of cancers, either as tumour promoters or tumour suppressors. Whilst Runx2 is renowned for its role as a master regulator of bone development we discuss here its expression pattern and putative functions beyond this lineage. Furthermore, we review the evidence that RUNX2 promotes neoplastic development in haematopoietic lineages and in advanced mammary and prostate cancer.

A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but no evidence of gammaC gene oncogenicity.

The emergence of leukemia following gene transfer to restore common cytokine receptor gamma chain (gammaC) function in X-linked severe combined immunodeficiency (SCID-X1) has raised important questions with respect to gene therapy safety. To explore the risk factors involved, we tested the oncogenic potential of human gammaC in new strains of transgenic mice expressing the gene under the control of the CD2 promoter and locus control region (LCR). These mice demonstrated mildly perturbed T-cell development, with an increased proportion of thymic CD8 cells, but showed no predisposition to tumor development even on highly tumor prone backgrounds or after gamma-retrovirus infection. The human CD2-gammaC transgene rescued T and B-cell development in gammaC(-/-) mice but with an age-related delay, mimicking postnatal reconstitution in SCID-X1 gene therapy subjects. However, we noted that gammaC(-/-) mice are acutely susceptible to murine leukemia virus (MLV) leukemogenesis, and that this trait was not corrected by the gammaC transgene. We conclude that the SCID-X1 phenotype can be corrected safely by stable ectopic expression of gammaC and that the transgene is not significantly oncogenic when expressed in this context. However, an underlying predisposition conferred by the SCID-X1 background appears to collaborate with insertional mutagenesis to increase the risk of tumor development.

Complex Interplay between the RUNX Transcription Factors and Wnt/ß-Catenin Pathway in Cancer: A Tango in the Night.

Cells are designed to be sensitive to a myriad of external cues so they can fulfil their individual destiny as part of the greater whole. A number of well-characterised signalling pathways dictate the cell's response to the external environment and incoming messages. In healthy, well-ordered homeostatic systems these signals are tightly controlled and kept in balance. However, given their powerful control over cell fate, these pathways, and the transcriptional machinery they orchestrate, are frequently hijacked during the development of neoplastic disease. A prime example is the Wnt signalling pathway that can be modulated by a variety of ligands and inhibitors, ultimately exerting its effects through the ß-catenin transcription factor and its downstream target genes. Here we focus on the interplay between the three-member family of RUNX transcription factors with the Wnt pathway and how together they can influence cell behaviour and contribute to cancer development. In a recurring theme with other signalling systems, the RUNX genes and the Wnt pathway appear to operate within a series of feedback loops. RUNX genes are capable of directly and indirectly regulating different elements of the Wnt pathway to either strengthen or inhibit the signal. Equally, ß-catenin and its transcriptional co-factors can control RUNX gene expression and together they can collaborate to regulate a large number of third party co-target genes.

Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy.

RUNX1 Is a Driver of Renal Cell Carcinoma Correlating with Clinical Outcome.

The recurring association of specific genetic lesions with particular types of cancer is a fascinating and largely unexplained area of cancer biology. This is particularly true of clear cell renal cell carcinoma (ccRCC) where, although key mutations such as loss of VHL is an almost ubiquitous finding, there remains a conspicuous lack of targetable genetic drivers. In this study, we have identified a previously unknown protumorigenic role for the RUNX genes in this disease setting. Analysis of patient tumor biopsies together with loss-of-function studies in preclinical models established the importance of RUNX1 and RUNX2 in ccRCC. Patients with high RUNX1 (and RUNX2) expression exhibited significantly poorer clinical survival compared with patients with low expression. This was functionally relevant, as deletion of RUNX1 in ccRCC cell lines reduced tumor cell growth and viability in vitro and in vivo. Transcriptional profiling of RUNX1-CRISPR-deleted cells revealed a gene signature dominated by extracellular matrix remodeling, notably affecting STMN3, SERPINH1, and EPHRIN signaling. Finally, RUNX1 deletion in a genetic mouse model of kidney cancer improved overall survival and reduced tumor cell proliferation. In summary, these data attest to the validity of targeting a RUNX1-transcriptional program in ccRCC. SIGNIFICANCE: These data reveal a novel unexplored oncogenic role for RUNX genes in kidney cancer and indicate that targeting the effects of RUNX transcriptional activity could be relevant for clinical intervention in ccRCC.

Runx1 promotes B-cell survival and lymphoma development.

Runx1 is essential for the homeostatic control of normal hematopoiesis and is required for lymphoid development. Translocations or point mutations that result in RUNX1 loss or disrupted function predispose to leukemia but data derived from model systems suggests that Runx genes can also be pro-oncogenic. Here we investigate the effects of enforced Runx1 expression in lymphoid lineages both in vivo and in vitro and show that transgene expression enhanced cell survival in the thymus and bone marrow but strongly inhibited the expansion of hematopoietic and B cell progenitors in vitro. Despite this, modestly enhanced levels of Runx1 accelerated Myc-induced lymphomagenesis in both the B cell and T cell lineages. Together these data provide formal proof that wild type Runx1 can promote oncogenesis in lymphoid tissues and that, in addition to loss of function, gain of function may have an aetiological role in leukemia.

Runx2 and MYC collaborate in lymphoma development by suppressing apoptotic and growth arrest pathways in vivo.

Members of the Runx and MYC families have been implicated as collaborating oncogenes. The mechanism of this potent collaboration is elucidated in this study of Runx2/MYC mice. As shown previously, ectopic expression of Runx2 in the thymus leads to a preneoplastic state defined by an accumulation of cells with an immature phenotype and a low proliferative rate. We now show that c-MYC overexpression is sufficient to rescue proliferation and to release the differentiation block imposed by Runx2. Analysis of Runx2-expressing lymphomas reveals a consistently low rate of apoptosis, in contrast to lymphomas of MYC mice which are often highly apoptotic. The low apoptosis phenotype is dominant in Runx2/MYC tumors, indicating that Runx2 confers a potent survival advantage to MYC-expressing tumor cells. The role of the p53 pathway in Runx2/MYC tumors was explored on a p53 heterozygote background. Surprisingly, functional p53 was retained in vivo, even after transplantation, whereas explanted tumor cells displayed rapid allele loss in vitro. Our results show that Runx2 and MYC overcome distinct "fail-safe" responses and that their selection as collaborating genes is due to their ability to neutralize each other's negative growth effect. Furthermore, the Runx2/MYC combination overcomes the requirement for genetic inactivation of the p53 pathway in vivo.

Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell lymphoma.

The RUNX1/AML1 gene is a frequent target for chromosomal translocations in human leukemia. The biological properties of the resulting fusion products and the finding that haploinsufficiency increases the risk of developing leukemia (W-J. Song et al., Nat. Genet., 23: 166-175, 1999; M. Osata et al., Blood, 93: 1817-1824, 1999) have led to the widely held view that RUNX1 loss-of-function is a key event. However, we now report that the gene is a target for insertional mutagenesis in T-cell lymphomas of mice carrying a MYC oncogene, where promoter insertion results in overexpression without affecting the integrity of the coding sequence. Moreover, Runx1 haploinsufficiency does not accelerate lymphoma development in MYC/Runx2 transgenic or murine leukemia virus-infected mice. These findings reveal that the Runx1 gene can also act as a dominant oncogene and suggest that the involvement of the Runx gene family in human leukemia may be more widespread and complex than previously realized.

Gamma-Retrovirus Integration Marks Cell Type-Specific Cancer Genes: A Novel Profiling Tool in Cancer Genomics.

Retroviruses have been foundational in cancer research since early studies identified proto-oncogenes as targets for insertional mutagenesis. Integration of murine gamma-retroviruses into the host genome favours promoters and enhancers and entails interaction of viral integrase with host BET/bromodomain factors. We report that this integration pattern is conserved in feline leukaemia virus (FeLV), a gamma-retrovirus that infects many human cell types. Analysis of FeLV insertion sites in the MCF-7 mammary carcinoma cell line revealed strong bias towards active chromatin marks with no evidence of significant post-integration growth selection. The most prominent FeLV integration targets had little overlap with the most abundantly expressed transcripts, but were strongly enriched for annotated cancer genes. A meta-analysis based on several gamma-retrovirus integration profiling (GRIP) studies in human cells (CD34+, K562, HepG2) revealed a similar cancer gene bias but also remarkable cell-type specificity, with prominent exceptions including a universal integration hotspot at the long non-coding RNA MALAT1. Comparison of GRIP targets with databases of super-enhancers from the same cell lines showed that these have only limited overlap and that GRIP provides unique insights into the upstream drivers of cell growth. These observations elucidate the oncogenic potency of the gamma-retroviruses and support the wider application of GRIP to identify the genes and growth regulatory circuits that drive distinct cancer types.

The Runx genes: lineage-specific oncogenes and tumor suppressors.

The Runx genes present a challenge to the simple binary classification of cancer genes as oncogenes or tumor suppressors. There is evidence that loss of function of two of the three mammalian Runx genes promotes cancer, but in a highly lineage-restricted manner. In human leukemias, the RUNX1 gene is involved in various chromosomal translocation events that create oncogenic fusion proteins, at least some of which appear to function as dominant-negative inhibitors of the normal gene product. Paradoxically, evidence is mounting that structurally intact Runx genes are also oncogenic when overexpressed. All the three murine genes act as targets for transcriptional activation by retroviral insertional mutagenesis, and the oncogenic potential of Runx2 has been confirmed in transgenic mice. Moreover, the RUNX1 gene is often amplified or overexpressed in cases of acute leukemia. The state of progress in elucidating the oncogenic roles of the Runx genes is the subject of this review, and we draw together recent observations in a tentative model for the effects of Runx deregulation on hematopoietic cell differentiation. We suggest that lineage-specific factors determine the sensitivity to the oncogenic effects of loss or overexpression of Runx factors.

RUNX1 transformation of primary embryonic fibroblasts is revealed in the absence of p53.

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19(ARF)-p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently pro-oncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFbeta indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

Runx2 contributes to the regenerative potential of the mammary epithelium.

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling.

Expression of RUNX1 correlates with poor patient prognosis in triple negative breast cancer.

The RUNX1 transcription factor is widely recognised for its tumour suppressor effects in leukaemia. Recently a putative link to breast cancer has started to emerge, however the function of RUNX1 in breast cancer is still unknown. To investigate if RUNX1 expression was important to clinical outcome in primary breast tumours a tissue microarray (TMA) containing biopsies from 483 patients with primary operable invasive ductal breast cancer was stained by immunohistochemistry. RUNX1 was associated with progesterone receptor (PR)-positive tumours (P<0.05), more tumour CD4+(P<0.05) and CD8+(P<0.01) T-lymphocytic infiltrate, increased tumour CD138+plasma cell (P<0.01) and more CD68+macrophage infiltrate (P<0.001). RUNX1 expression did not influence outcome of oestrogen receptor (ER)-positive or HER2-positive disease, however on univariate analysis a high RUNX1 protein was significantly associated with poorer cancer-specific survival in patients with ER-negative (P<0.05) and with triple negative (TN) invasive breast cancer (P<0.05). Furthermore, multivariate Cox regression analysis of cancer-specific survival showed a trend towards significance in ER-negative patients (P<0.1) and was significant in triple negative patients (P<0.05). Of relevance, triple negative breast cancer currently lacks good biomarkers and patients with this subtype do not benefit from the option of targeted therapy unlike patients with ER-positive or HER2-positive disease. Using multivariate analysis RUNX1 was identified as an independent prognostic marker in the triple negative subgroup. Overall, our study identifies RUNX1 as a new prognostic indicator correlating with poor prognosis specifically in the triple negative subtype of human breast cancer.