RUNX transcription factors: biological functions and implications in cancer.

Runt-related transcription factors (RUNX) are a family of transcription factors that are essential for normal and malignant hematopoietic processes. Their most widely recognized role in malignancy is to promote the occurrence and development of acute myeloid leukemia. However, it is worth noting that during the last decade, studies of RUNX proteins in solid tumors have made considerable progress, suggesting that these proteins are directly involved in different stages of tumor development, including tumor initiation, progression, and invasion. RUNX proteins also play a role in tumor angiogenesis, the maintenance of tumor cell stemness, and resistance to antitumor drugs. These findings have led to the consideration of RUNX as a tumor biomarker. All RUNX proteins are involved in the occurrence and development of solid tumors, but the role of each RUNX protein in different tumors and the major signaling pathways involved are complicated by tumor heterogeneity and the interacting tumor microenvironment. Understanding how the dysregulation of RUNX in tumors affects normal biological processes is important to elucidate the molecular mechanisms by which RUNX affects malignant tumors.

Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms.

Runx factors are essential for lineage specification of various hematopoietic cells, including T lymphocytes. However, they regulate context-specific genes and occupy distinct genomic regions in different cell types. Here, we show that dynamic Runx binding shifts in mouse early T cell development are mostly not restricted by local chromatin state but regulated by Runx dosage and functional partners. Runx cofactors compete to recruit a limited pool of Runx factors in early T progenitor cells, and a modest increase in Runx protein availability at pre-commitment stages causes premature Runx occupancy at post-commitment binding sites. This increased Runx factor availability results in striking T cell lineage developmental acceleration by selectively activating T cell-identity and innate lymphoid cell programs. These programs are collectively regulated by Runx together with other, Runx-induced transcription factors that co-occupy Runx-target genes and propagate gene network changes.

The RUNX Family of Proteins, DNA Repair, and Cancer.

The RUNX family of transcription factors, including RUNX1, RUNX2, and RUNX3, are key regulators of development and can function as either tumor suppressors or oncogenes in cancer. Emerging evidence suggests that the dysregulation of RUNX genes can promote genomic instability in both leukemia and solid cancers by impairing DNA repair mechanisms. RUNX proteins control the cellular response to DNA damage by regulating the p53, Fanconi anemia, and oxidative stress repair pathways through transcriptional or non-transcriptional mechanisms. This review highlights the importance of RUNX-dependent DNA repair regulation in human cancers.

RUNX3 in Stem Cell and Cancer Biology.

The runt-related transcription factors (RUNX) play prominent roles in cell cycle progression, differentiation, apoptosis, immunity and epithelial-mesenchymal transition. There are three members in the mammalian RUNX family, each with distinct tissue expression profiles. RUNX genes play unique and redundant roles during development and adult tissue homeostasis. The ability of RUNX proteins to influence signaling pathways, such as Wnt, TGFß and Hippo-YAP, suggests that they integrate signals from the environment to dictate cell fate decisions. All RUNX genes hold master regulator roles, albeit in different tissues, and all have been implicated in cancer. Paradoxically, RUNX genes exert tumor suppressive and oncogenic functions, depending on tumor type and stage. Unlike RUNX1 and 2, the role of RUNX3 in stem cells is poorly understood. A recent study using cancer-derived RUNX3 mutation R122C revealed a gatekeeper role for RUNX3 in gastric epithelial stem cell homeostasis. The corpora of RUNX3R122C/R122C mice showed a dramatic increase in proliferating stem cells as well as inhibition of differentiation. Tellingly, RUNX3R122C/R122C mice also exhibited a precancerous phenotype. This review focuses on the impact of RUNX3 dysregulation on (1) stem cell fate and (2) the molecular mechanisms underpinning early carcinogenesis.

The RUNX Family, a Novel Multifaceted Guardian of the Genome.

The DNA repair machinery exists to protect cells from daily genetic insults by orchestrating multiple intrinsic and extrinsic factors. One such factor recently identified is the Runt-related transcription factor (RUNX) family, a group of proteins that act as a master transcriptional regulator for multiple biological functions such as embryonic development, stem cell behaviors, and oncogenesis. A significant number of studies in the past decades have delineated the involvement of RUNX proteins in DNA repair. Alterations in RUNX genes cause organ failure and predisposition to cancers, as seen in patients carrying mutations in the other well-established DNA repair genes. Herein, we review the currently existing findings and provide new insights into transcriptional and non-transcriptional multifaceted regulation of DNA repair by RUNX family proteins.

RUNX Proteins as Epigenetic Modulators in Cancer.

RUNX proteins are highly conserved in metazoans and perform critical functions during development. Dysregulation of RUNX proteins through various molecular mechanisms facilitates the development and progression of various cancers, where different RUNX proteins show tumor type-specific functions and regulate different aspects of tumorigenesis by cross-talking with different signaling pathways such as Wnt, TGF-ß, and Hippo. Molecularly, they could serve as transcription factors (TFs) to activate their direct target genes or interact with many other TFs to modulate chromatin architecture globally. Here, we review the current knowledge on the functions and regulations of RUNX proteins in different cancer types and highlight their potential role as epigenetic modulators in cancer.

RUNX Family in Hypoxic Microenvironment and Angiogenesis in Cancers.

The tumor microenvironment (TME) is broadly implicated in tumorigenesis, as tumor cells interact with surrounding cells to influence the development and progression of the tumor. Blood vessels are a major component of the TME and are attributed to the creation of a hypoxic microenvironment, which is a common feature of advanced cancers and inflamed premalignant tissues. Runt-related transcription factor (RUNX) proteins, a transcription factor family of developmental master regulators, are involved in vital cellular processes such as differentiation, proliferation, cell lineage specification, and apoptosis. Furthermore, the RUNX family is involved in the regulation of various oncogenic processes and signaling pathways as well as tumor suppressive functions, suggesting that the RUNX family plays a strategic role in tumorigenesis. In this review, we have discussed the relevant findings that describe the crosstalk of the RUNX family with the hypoxic TME and tumor angiogenesis or with their signaling molecules in cancer development and progression.

The Roles of RUNX Proteins in Lymphocyte Function and Anti-Tumor Immunity.

The Runt-related transcription factor (RUNX) family of proteins are crucial for many developmental and immuno-physiological processes. Their importance in cellular and tissue development has been repeatedly demonstrated as they are often found mutated and implicated in tumorigenesis. Most importantly, RUNX have now emerged as critical regulators of lymphocyte function against pathogenic infections and tumorigenic cells, the latter has now revolutionized our current understandings as to how RUNX proteins contribute to control tumor pathogenicity. These multifunctional roles of RUNX in mammalian immune responses and tissue homeostasis have led us to appreciate their value in controlling anti-tumor immune responses. Here, we summarize and discuss the role of RUNX in regulating the development and function of lymphocytes responding to foreign and tumorigenic threats and highlight their key roles in anti-tumor immunity.

Failure of human rhombic lip differentiation underlies medulloblastoma formation.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

Runx proteins and transcriptional mechanisms that govern memory CD8 T cell development.

Adaptive immunity to intracellular pathogens and tumors is mediated by antigen-experienced CD8 T cells. Individual naive CD8 T cells have the potential to differentiate into a diverse array of antigen-experienced subsets that exhibit distinct effector functions, life spans, anatomic positioning, and potential for regenerating an entirely new immune response during iterative pathogenic exposures. The developmental process by which activated naive cells undergo diversification involves regulation of chromatin structure and transcription but is not entirely understood. This review examines how alterations in chromatin structure, transcription factor binding, extracellular signals, and single-cell gene expression explain the differential development of distinct effector (TEFF ) and memory (TMEM ) CD8 T cell subsets. Special emphasis is placed on how Runx proteins function with additional transcription factors to pioneer changes in chromatin accessibility and drive transcriptional programs that establish the core attributes of cytotoxic T lymphocytes, subdivide circulating and non-circulating TMEM cell subsets, and govern terminal differentiation. The discussion integrates the roles of specific cytokine signals, transcriptional circuits and how regulation of individual nucleosomes and RNA polymerase II activity can contribute to the process of differentiation. A model that integrates many of these features is discussed to conceptualize how activated CD8 T cells arrive at their fates.

Gene expression patterns associated with dental replacement in the rabbit, a new model for the mammalian dental replacement mechanisms.

BACKGROUND: Unlike many vertebrates with continuous dental replacement, mammals have a maximum of two dental generations. Due to the absence of dental replacement in the laboratory mouse, the mechanisms of the mammalian tooth replacement system are poorly known. In this study, we use the European rabbit as a model for mammalian tooth development and replacement. RESULTS: We provide data on some key regulators of tooth development. We detected the presence of SOX2 in both the replacement dental lamina and the rudimentary successional dental lamina of unreplaced molars, indicating that SOX2 may not be sufficient to initiate and maintain tooth replacement. We showed that Shh does not seem to be directly involved in tooth replacement. The transient presence of the rudimentary successional dental lamina in the molar allowed us to identify genes that could be essential for the initiation or the maintenance of tooth replacement. Hence, the locations of Sostdc1, RUNX2, and LEF1 vary between the deciduous premolar, the replacement premolar, and the molar, indicating possible roles in tooth replacement. CONCLUSION: According to our observations, initiation and the maintenance of tooth replacement correlate with the presence of LEF1+ cells and the absence of both mesenchymal RUNX2 and epithelial Sostdc1+ cells.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Tissue Engineering of Cartilage Using a Random Positioning Machine.

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.

Glycogen synthase kinase-3 promotes T helper type 17 differentiation by promoting interleukin-9 production.

T helper type 17 (Th17) cells are recognized as important contributors to the deleterious effects of several neurological and psychiatric diseases. Clarifying mechanisms that control the production of Th17 cells may therefore provide new strategies for developing novel interventions in a broad spectrum of disorders. Th17 cell differentiation is promoted by glycogen synthase kinase-3 (GSK3), but the mechanisms for this are only beginning to be understood. Using T-cell-selective depletion of GSK3ß and multiple selective pharmacological GSK3 inhibitors, we found that GSK3 inhibition decreased C-C motif chemokine (ccl)20, C-C motif chemokine receptor (ccr)6, interleukin (IL)-9, Runt-related transcription factor (Runx)1, interferon regulatory factor (Irf)4 and c-maf mRNA expression after 2 days of Th17 cell differentiation in vitro. These effects were found to be independent of the master regulator transcription factor retinoic acid receptor-related orphan receptor γT (RORγT), as GSK3 inhibition still reduced Th17 cell differentiation in RORγT-depleted cells. Because IL-9 was approximately ninefold down-regulated in GSK3ß-/- CD4 cells, we tested if reintroduction of IL-9 during Th17 cell differentiation abolished the inhibition by GSK3 deficiency of Th17 cell differentiation. We found that IL-9 over-expression was sufficient to reverse the inhibition of Th17 cell differentiation by GSK3 inhibition or depletion. We found that IL-9 enhances Th17 cell differentiation in part through signal transducer and activator of transcription 3 (STAT3) activation, and IL-9 also enhances STAT3 binding to the IL-17a promoter. Altogether, these findings suggest that IL-9 might be an important mediator of GSK3ß-dependent enhancement of Th17 cell differentiation.

Role of RUNX Family Transcription Factors in DNA Damage Response.

Cells are constantly exposed to endogenous and exogenous stresses that can result in DNA damage. In response, they have evolved complex pathways to maintain genomic integrity. RUNX family transcription factors (RUNX1, RUNX2, and RUNX3 in mammals) are master regulators of development and differentiation, and are frequently dysregulated in cancer. A growing body of research also implicates RUNX proteins as regulators of the DNA damage response, often acting in conjunction with the p53 and Fanconi anemia pathways. In this review, we discuss the functional role and mechanisms involved in RUNX factor mediated response to DNA damage and other cellular stresses. We highlight the impact of these new findings on our understanding of cancer predisposition associated with RUNX factor dysregulation and their implications for designing novel approaches to prevent cancer formation in affected individuals.

CROX (Cluster Regulation of RUNX) as a Potential Novel Therapeutic Approach.

Comprehensive inhibition of RUNX1, RUNX2, and RUNX3 led to marked cell suppression compared with inhibition of RUNX1 alone, clarifying that the RUNX family members are important for proliferation and maintenance of diverse cancers, and "cluster regulation of RUNX (CROX)" is a very effective strategy to suppress cancer cells. Recent studies reported by us and other groups suggested that wild-type RUNX1 is needed for survival and proliferation of certain types of leukemia, lung cancer, gastric cancer, etc. and for their one of metastatic target sites such as born marrow endothelial niche, suggesting that RUNX1 often functions oncogenic manners in cancer cells. In this review, we describe the significance and paradoxical requirement of RUNX1 tumor suppressor in leukemia and even solid cancers based on recent our findings such as "genetic compensation of RUNX family transcription factors (the compensation mechanism for the total level of RUNX family protein expression)", "RUNX1 inhibition-induced inhibitory effects on leukemia cells and on solid cancers through p53 activation", and "autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells". Taken together, these findings identify a crucial role for the RUNX cluster in the maintenance and progression of cancers and suggest that modulation of the RUNX cluster using the pyrrole-imidazole polyamide gene-switch technology is a potential novel therapeutic approach to control cancers.

Role of RUNX Family Members in G1 Restriction-Point Regulation.

When cells are stimulated by growth factors, they make a critical choice in early G1 phase: proceed forward to S phase, remain in G1, or revert to G0 phase. Once the critical decision is made, cells execute a fixed program independently of extracellular signals. The specific stage at which the critical decision is made is called the restriction point or R-point. The existence of the R-point raises a major question: what is the nature of the molecular machinery that decides whether or not a cell in G1 will continue to advance through the cell cycle or exit from the cell cycle? The R-point program is perturbed in nearly all cancer cells. Therefore, exploring the nature of the R-point decision-making machinery will provide insight into how cells consult extracellular signals and intracellular status to make an appropriate R-point decision, as well into the development of cancers. Recent studies have shown that expression of a number of immediate early genes is associated with the R-point decision, and that the decision-making program constitutes an oncogene surveillance mechanism. In this review, we briefly summarize recent findings regarding the mechanisms underlying the context-dependent R-point decision.

The Roles of RUNX Family Proteins in Development of Immune Cells.

The Runt-related transcription factors (RUNX) transcription factors have been known for their critical roles in numerous developmental processes and diseases such as autoimmune disorders and cancer. Especially, RUNX proteins are best known for their roles in hematopoiesis, particularly during the development of T cells. As scientists discover more types of new immune cells, the functional diversity of RUNX proteins also has been increased over time. Furthermore, recent research has revealed complicated transcriptional networks involving RUNX proteins by the current technical advances. Databases established by next generation sequencing data analysis has identified ever increasing numbers of potential targets for RUNX proteins and other transcription factors. Here, we summarize diverse functions of RUNX proteins mainly on lymphoid lineage cells by incorporating recent discoveries.

Involvement of RUNX and BRD Family Members in Restriction Point.

A tumor is an abnormal mass of tissue that arises when cells divide more than they should or do not die when they should. The cellular decision regarding whether to undergo division or death is made at the restriction (R)-point. Consistent with this, an increasingly large body of evidence indicates that deregulation of the R-point decision-making machinery accompanies the formation of most tumors. Although the R-point decision is literally a matter of life and death for the cell, and thus critical for the health of the organism, it remains unclear how a cell chooses its own fate. Recent work demonstrated that the R-point constitutes a novel oncogene surveillance mechanism operated by R-point-associated complexes of which RUNX3 and BRD2 are the core factors (Rpa-RX3 complexes). Here, we show that not only RUNX3 and BRD2, but also other members of the RUNX and BRD families (RUNX1, RUNX2, BRD3, and BRD4), are involved in R-point regulation.

RUNX transcription factors: orchestrators of development.

RUNX transcription factors orchestrate many different aspects of biology, including basic cellular and developmental processes, stem cell biology and tumorigenesis. In this Primer, we introduce the molecular hallmarks of the three mammalian RUNX genes, RUNX1, RUNX2 and RUNX3, and discuss the regulation of their activities and their mechanisms of action. We then review their crucial roles in the specification and maintenance of a wide array of tissues during embryonic development and adult homeostasis.