• 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.
    • 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.
    • Targeting the translational machinery as a novel treatment strategy for non-Hodgkin lymphoma

      Hagner, Patrick; Gartenhaus, Ronald B.; Passaniti, Antonino (2010)
      Non-Hodgkin lymphoma (NHL) is a heterogeneous group of B-cell and T-cell malignancies, which originate in tissues of the lymph system. According to the National Cancer Institute, non-Hodgkin lymphoma is the fifth most diagnosed cancer in the country. Diffuse large B-cell lymphoma is the most common of the NHL, accounting for approximately 40% of all NHL. Although the transcriptional mechanisms involving c-MYC and BCL6 have been extensively studied in DLBCL, little is known about the role of post-transcriptional mechanisms involved in lymphomagenesis. The dysregulation of protein synthesis evident in the transformed phenotype has opened up a burgeoning field of research in cancer biology. Translation initiation has recently been shown to be a common downstream target of signal transduction pathways deregulated in cancer and initiated by mutated/overexpressed oncogenes and tumor suppressors. The overexpression and/or activation of proteins involved in translation initiation such as eIF4E, mTOR and eIF4G have been shown to induce a malignant phenotype. Therefore, understanding the mechanisms that control protein synthesis is emerging as an exciting new research area with significant potential for developing innovative therapies. We demonstrated that the oncogene MCT-1 is highly expressed in 85% of primary DLBCL samples. Disruption of MCT-1 function by either a dominant negative or inhibitory RNA (RNAi) attenuated the malignant phenotype through a mechanism involving the modulation of the translational profile present in the lymphoma. Microarray analysis of actively translating polysomes demonstrated that inhibition of MCT-1 led to an increase in the translation of messages with tumor suppressor functions and a decrease in the translation of oncogenic messages. The inverse correlation between chronic alcohol consumption and incidence of NHL has been observed in multiple epidemiological studies. We demonstrated that ethanol inhibits mammalian target of rapamycin (mTOR) signaling, which resulted in a decrease in cap-dependent translation. Microarray analysis of mRNA bound to actively translating ribosomes demonstrated those messages most significantly repressed encode the translational machinery. Further, chronic ethanol exposure resulted in diminished tumor formation in a pre-clinical human lymphoma xenograft model. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins. We demonstrated that ribosomal protein S6 (RPS6) is highly expressed in primary DLBCL samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lead to an increase in the apoptotic population of cells compared to control shRNA. We also demonstrated that RPS6 regulates the translation of messages containing a 5' terminal oligopyrimidine (5' TOP) tract, which encode the translational machinery. These findings are significant for the clinical setting as specifically targeting molecules upon which DLBCL depends for the survival of the disease may achieve more significant patient responses. Therefore we believe that targeting the translational machinery may constitute a novel therapeutic approach in the treatment of cancer.
    • Tumor Angiogenesis and the role of RUNX2 transcription factor in endothelial cell function: regulation by glucose levels and oxidative stress

      Kommineni, Sravya; Passaniti, Antonino (2011)
      The RUNX2 DNA-binding transcription factor is an important regulator of tumor angiogenesis. Its transcriptional activity is dependent on interaction with a cofactor, CBF?, which does not bind DNA, but enhances RUNX2 DNA binding. Recently, we discovered that RUNX2 DNA binding activity is regulated by glucose and the redox status of the cell. Euglycemic levels of glucose initiate a series of events leading to RUNX2 phosphorylation and DNA binding. In the presence of elevated levels of glucose (hyperglycemia), RUNX2 oxidation may reduce its ability to bind DNA. Therefore, we propose the hypothesis that the RUNX2 transcription factor regulates endothelial cell (EC) proliferation through its glucose response and redox status. To test this hypothesis, in specific aim 1, we will examine the ability of RUNX2 to associate with a target gene that regulates cell cycle progression and proliferation, the p21Cip1 promoter. This DNA interaction will be examined by chromatin immunoprecipitation assays and by DNA-binding assays. In specific aim 2, we will examine the effect of mutations in critical cysteine and methionine residues within RUNX2 that regulate DNA binding. The effect of these mutations on DNA binding will be examined using electrophoretic mobility shift assays. Two mutant proteins will be used: a GFP.tagged RUNT DNA binding domain fusion protein in which a methionine is converted to a valine (RUNT.M106V) and a cysteine to serine substitution in full-length RUNX2 at position 118 (RUNX2.C118S). These studies will help define the mechanisms through which nutrients and cellular redox status regulate RUNX2 activity and will provide opportunities for new therapeutic approaches.