• Characterization of the role of the RUNX2 transcriptional regulator in angiogenesis and oncogenic transformation

      Vitolo, Michele I.; Passaniti, Antonino (2004)
      Carcinogenesis is a complex process that is regulated by a variety of host responses, which include neovascularization, or angiogenesis. Vascular endothelial cell (EC) proliferation, migration, and tissue invasion are critical for the establishment of new blood vessels within tumors. These EC functions are under the control of key transcriptional regulators that modulate the expression of angiogenic factors, cytokine receptors, and proteases. The expression and functional role of the transcription factor runt-related gene 2 (RUNX2) has been implicated in a variety of biological and pathological events. The aim of this thesis is to begin to characterize the role of RUNX2 in angiogenesis and oncogenic transformation. A major finding of this study indicates that RUNX2 regulates cell growth arrest, differentiation, apoptosis, and migration of endothelial cells exposed to extracellular matrix (ECM). Each of these biological functions is a critical step in the complex process of angiogenesis. This work has also elucidated a novel function for a naturally occurring splice-variant of RUNX2 that was determined to be a competitive inhibitor of RUNX2, lending further support to the hypothesis that ECs regulate growth and apoptosis through RUNX2 alternative splicing events. Further studies using cultured fibroblasts supported a role for oncogenic transformation through interaction with the YES-associated transcriptional coactivator, YAP. Elucidating the basic mechanisms of RUNX2 gene regulation in the process of angiogenesis may provide potential pharmacological approaches to inhibit angiogenesis and prevent tumor growth and metastasis. Accordingly, determining the mechanism of RUNX2 in oncogenic transformation may similarly lead to novel therapeutic approaches to directly target tumor cells and inhibit the progression of cancer.
    • The intersecting function of Connexin 43 and Runx2 in bone

      Buo, Atum Michael Lionel; Stains, Joseph P.; 0000-0001-9610-2623 (2017)
      Bone is formed and maintained through the tightly coordinated activities of osteoblasts, osteoclasts, and osteocytes. Connexin 43 (Cx43), the most predominantly expressed gap junction protein in bone, facilitates the coordination of bone cell function via the gap junction-mediated sharing of second messengers throughout the osteoblast-osteocyte network. However, the underlying mechanistic details explaining how Cx43 converts shared second messengers into signals that induce essential osteogenic processes are largely unclear. Therefore, this work investigates whether Cx43 impacts osteoblast differentiation and bone quality by regulating Runx2, the master transcriptional regulator of osteogenesis. To assess this in vitro, we devised a reproducible Cre/LoxP-based system to delete the Cx43 gene (Gja1) from murine primary osteoblasts, and hypothesized that adenoviral overexpression of Runx2 in these cells would be able to rescue the defective osteogenesis caused by loss of Cx43. To assess this in vivo, we used a compound hemizygous breeding strategy to generate mice that are doubly hemizygous for the Cx43 gene (Gja1) and the Runx2 gene (Cx43+/- Runx2+/-), and then utilized micro-CT scanning on femurs and skulls to analyze the skeletal phenotype at 8 weeks of age. We hypothesized that if Cx43 and Runx2 indeed functionally intersect to regulate osteogenesis in vivo, then the dual hemizygosity of both Cx43 and Runx2 should manifest a skeletal phenotype not visible in wild-type or singly hemizygous animals. Our findings reveal that overexpressing Runx2 in Cx43-depleted cells rescues osteoblast differentiation by restoring osteocalcin gene expression and reducing proliferation. Additionally, cortical bones of compound Gja1+/- Runx2+/- mice, in comparison to wildtype and singly Gja1+/- and Runx2+/- littermate controls, have a marked increase in tissue area, a widened marrow cavity due to increased bone resorption, and a striking increase in porosity. Furthermore, the compound mice display cranial defects not observable in the other genotypes, and the calvarial osteoblasts from the compound mice also possess a hyperproliferative defect. Together, these findings strongly indicate that Cx43 and Runx2 functionally intersect to regulate osteogenesis in vitro and in vivo and provide greater insight towards understanding how Cx43 impacts bone quality.
    • An oncogenic role for RUNX2 in breast cancer progression and glucose metabolism through regulation of SIRT6

      Choe, Moran; Passaniti, Antonino (2013)
      Breast cancer (BC) progression is characterized by silencing of differentiation-specific genes and activation of genes promoting a switch from oxidative phosphorylation to aerobic glycolysis - the Warburg effect. Although the RUNX2 transcription factor promotes BC metastasis to bone, the mechanisms through which it regulates oncogenesis are not clear. We find that RUNX2 induction in MCF7 cells is associated with reduction in differentiation-specific estrogen receptor-α (ERα), increased expression of several glycolytic genes, increased glucose uptake (GLUT1), sensitivity to glucose starvation, and resistance to mitochondrial oxidative phosphorylation (OXPHOS) inhibitors. Conversely, endogenous RUNX2 knockdown in Hs578t triple-negative BC cells reduced cellular glucose addiction and expression of several glycolytic genes. Interestingly, these cells maintained high levels of PDHA1, which promotes OXPHOS by converting pyruvate to acetyl CoA to enter TCA cycle. In addition, RUNX2 knockdown resulted in a significant increase in oxygen consumption rate (OCR), indicative of enhanced mitochondrial OXPHOS. Mechanistically, the NAD-dependent histone deacetylase SIRT6, a known tumor suppressor, was a critical regulator of the RUNX2-mediated metabolic switch. SIRT6 levels were reduced in malignant BC tissues or cell lines that expressed high levels of RUNX2. This repression was regulated at both the transcriptional and post-translational levels and may account for the glycolytic phenotype and reduced mitochondrial OXPHOS in RUNX2 positive BC cells. Moreover, the expression of pyruvate dehydrogenase kinase 1 (PDHK1), which phosphorylates and inactivates PDH, was significantly higher in RUNX2 positive cells relative to RUNX2 negative cells. This reduction of PDHK1 was also observed in SIRT6 overexpressing cells, which suggests a potential role for SIRT6 as a repressor of this oncogenic kinase. Finally, ectopic expression of SIRT6 in RUNX2 positive cells increased, while specific knockdown of SIRT6 in RUNX2 negative cells decreased, respiration. In summary, these results suggest that RUNX2-mediated repression of the SIRT6 tumor suppressor may be a key pathway that promotes the Warburg effect and BC tumor progression.
    • Regulation of glucose-activated RUNX2 phosphorylation: effects on endothelial cell proliferation and an angiogenic phenotype

      Pierce, Adam Daniel; Passaniti, Antonino (2011)
      The runt-related protein-2 (RUNX2) is a DNA-binding transcription factor that regulates bone formation, tumor cell metastasis, endothelial cell (EC) proliferation, and angiogenesis. RUNX2 DNA binding is glucose and cell cycle regulated. We propose that glucose may activate RUNX2 through changes in post-translational phosphorylation that are cell cycle-specific and will regulate EC function. Glucose increased cell cycle progression in EC through both G2/M and G1 phases with entry into S-phase occurring only in subconfluent cells. In the absence of nutrients and growth factors (starvation), subconfluent EC were delayed in G1 when RUNX2 expression was reduced. RUNX2 phosphorylation, activation of DNA binding, and pRb phosphorylation were stimulated by glucose and were necessary to promote cell cycle progression. Glucose increased RUNX2 localization at focal subnuclear sites, which co-incided with RUNX2 occupancy of the cyclin-dependent kinase (cdk) inhibitor p21Cip1 promoter, a gene normally repressed by RUNX2. Mutation of the RUNX2 cdk phosphorylation site in the C-terminal domain (S451A.RUNX2) reduced RUNX2 phosphorylation and DNA binding. Expression of this cdk site mutant in EC inhibited glucose-stimulated differentiation, monolayer wound healing, and proliferation. We also found that the methionine and cysteine residues of RUNX2 were sensitive to reactive oxygen species (ROS) that directly regulated RUNX2 DNA-binding activity in ECs. Functional screening of RUNX2-specific inhibitors identified the vitamin D3 prohormone to have a novel role in EC proliferation mediated through the regulation of RUNX2 activity. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data also suggest that regulation of RUNX2 DNA-binding activity by vitamin D3 or ROS may be useful to inhibit EC proliferation in tumor angiogenesis.
    • A Role For RUNX2 And TAZ In Promoting A Tumorigenic Phenotype In Luminal Breast Cancer Cells

      Brusgard, Jessica; Passaniti, Antonino (2014)
      Breast cancer (BC) is the second leading cause of cancer-associated deaths among women. Current strategies aimed at eradicating the primary tumor by targeting the bulk population of cells often leads to BC recurrence and metastasis because of intratumoral heterogeneity and treatment resistance. The RUNX2 transcription factor is upregulated in early stage luminal BC and is a poor prognostic indicator of patient survival. However, the precise mechanisms by which RUNX2 regulates an oncogenic phenotype in early stage tumors are not known. We now show for the first time that RUNX2 promotes luminal BC cell tumorsphere formation, which was inhibited with a novel RUNX2-targeting drug. RUNX2 associated with the TAZ transcriptional coactivator in MCF7 cells to promote a tumorigenic phenotype that was inhibited by siRNA-targeted knockdown of TAZ. TGFβ treatment of cells expressing RUNX2 increased endogenous TAZ translocation to the nucleus, a process that was prevented by inhibiting RUNX2. This translocation was preceded by disruption of adherens junctions through ectodomain shedding of an oncogenic soluble E-Cadherin fragment (80kDa sE-Cad). RUNX2 expression increased HER2-mediated tumorsphere formation, which was abrogated after treatment with the HER2-targeting agents Herceptin and Lapatinib. These data support a novel role for RUNX2 in promoting early stage tumorigenesis in the context of TGFβ, the Hippo signaling mediator TAZ, sE-Cad, and HER2. Using this signaling pathway to monitor BC cell oncogenic activity will allow us to discover new anti-cancer agents. Further characterization of RUNX2-targeted compounds that disrupt this oncogenic pathway could have therapeutic potential.