Kara C. Sedoris

c-Myc and Cancer Metabolism

The processes of cellular growth regulation and cellular metabolism are closely interrelated. The c-Myc oncogene is a “master regulator” which controls many aspects of both of these processes. The metabolic changes which occur in transformed cells, many of which are driven by c-Myc overexpression, are necessary to support the increased need for nucleic acids, proteins, and lipids necessary for rapid cellular proliferation. At the same time, c-Myc overexpression results in coordinated changes in level of expression of gene families which result in increased cellular proliferation. This interesting duality of c-Myc effects places it in the mainstream of transformational changes and gives it a very important role in regulating the “transformed phenotype.” The effects induced by c-Myc can occur either as a “primary oncogene” which is activated by amplification or translocation or as a downstream effect of other activated oncogenes. In either case, it appears that c-Myc plays a central role in sustaining the changes which occur with transformation. Although efforts to use c-Myc as a therapeutic target have been quite frustrating, it appears that this may change in the next few years. Clin Cancer Res; 18(20); 5546–53. ©2012 AACR.

Lung ischemia-reperfusion injury: implications of oxidative stress and platelet-arteriolar wall interactions.

Abstract Pulmonary ischemia–reperfusion (IR) injury may result from trauma, atherosclerosis, pulmonary embolism, pulmonary thrombosis and surgical procedures such as cardiopulmonary bypass and lung transplantation. IR injury induces oxidative stress characterized by formation of reactive oxygen (ROS) and reactive nitrogen species (RNS). Nitric oxide (NO) overproduction via inducible nitric oxide synthase (iNOS) is an important component in the pathogenesis of IR. Reaction of NO with ROS forms RNS as secondary reactive products, which cause platelet activation and upregulation of adhesion molecules. This mechanism of injury is particularly important during pulmonary IR with increased iNOS activity in the presence of oxidative stress. Platelet–endothelial interactions may play an important role in causing pulmonary arteriolar vasoconstriction and post-ischemic alveolar hypoperfusion. This review discusses the relationship between ROS, RNS, P-selectin, and platelet–arteriolar wall interactions and proposes a hypothesis for their role in microvascular responses during pulmonary IR.

Differential effects of nitric oxide synthesis on pulmonary vascular function during lung ischemia-reperfusion injury.

Lung ischemia-reperfusion (IR) injury causes alveolar, epithelial and endothelial cell dysfunction which often results in decreased alveolar perfusion, characteristic of an acute respiratory distress syndrome. Nitric oxide (NO) from endothelium-derived NO synthase (eNOS) helps maintain a low pulmonary vascular resistance. Paradoxically, during acute lung injury, overproduction of NO via inducible NO synthase (iNOS) and oxidative stress lead to reactive oxygen and nitrogen species (ROS and RNS) formation and vascular dysfunction. RNS potentiate vascular and cellular injury by oxidation, by decreasing NO bioavailability, and by regulating NOS isoforms. RNS potentiate their own production by uncoupling NO production through eNOS by oxidation and disruption of Akt-mediated phosphorylation of eNOS. This review focuses on effects of NO which cause vascular dysfunction in the unique environment of the lung and presents a hypothesis for interplay between eNOS and iNOS activation with implications for development of new strategies to treat vascular dysfunction associated with IR.

Genomic c-Myc Quadruplex DNA Selectively Kills Leukemia

c-Myc, a key regulator of cell cycle and proliferation, is commonly overexpressed in leukemia and associated with poor prognosis. Conventional antisense oligonucleotides targeting c-myc may attenuate leukemic cell growth, however, are poorly taken into cells, rapidly degraded, and have unwanted effects on normal cells. The c-myc promoter contains a guanine-rich sequence (PU27) capable of forming quadruplex (four-stranded) DNA, which may negatively regulate c-myc transcription. However, its biological significance is unknown. We show that treatment of leukemia with an oligonucleotide encoding the genomic PU27 sequence induces cell-cycle arrest and death by oncotic necrosis due to PU27-mediated suppression of c-myc mRNA/protein expression. Furthermore, PU27 is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on normal cells. Suppression of c-myc expression by PU27 caused significant DNA damage, cell and mitochondrial swelling, and membrane permeability characteristic of oncotic necrosis. Induction of oncosis caused mitochondrial dysfunction, depletion of cellular ATP levels, and enhanced oxidative stress. This novel antileukemic strategy addresses current concerns of oligonucleotide therapeutics including problems with uptake, stability, and unintentional effects on normal cells and is the first report of selective cancer cell killing by a genomic DNA sequence. Mol Cancer Ther; 11(1); 66–76. ©2011 AACR.

Interplay of endothelial and inducible nitric oxide synthases modulates the vascular response to ischaemia-reperfusion in the rabbit lung.

Aim:  Lung ischaemia–reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia–reperfusion‐induced pulmonary vasoconstriction. The aim was to determine whether ischaemia–reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion.

Abstract 3: Induction of mitochondrial autophagy in leukemia by the Bcl-2 quadruplex-forming sequence

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Bcl-2 (B-cell CLL/lymphoma 2), a mitochondrial membrane oncoprotein functioning as an inhibitor of apoptosis, is commonly overexpressed in a variety of hematological malignancies promoting apoptotic resistance. Inhibition of Bcl-2 may result in sustained regression of leukemia/lymphoma. The promoters of several cancer-related oncogenes, including Bcl-2, contain sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The Bcl-2 quadruplex-forming sequence (Bcl-2 q, 23 bp) is located upstream of the P1 promoter of the Bcl-2 gene and is implicated in negative regulation of Bcl-2 transcription, however, the biological role of this sequence remains unclear. We hypothesize that treatment of leukemia cells with an oligonucleotide encoding Bcl-2 q induces cell death by inhibiting Bcl-2 gene expression. To determine the biological role of the Bcl-2 q on leukemic cell proliferation, U937 cells were treated with Bcl-2 q or the corresponding mutant sequence (MutBcl-2), which lacks runs of two or more guanines. Our results demonstrate that Bcl-2 q formed a stable parallel quadruplex structure and showed remarkable serum and intracellular stability. Treatment of leukemia cells with Bcl-2 q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM), which was not a result of cell cycle arrest. No change in growth of non-transformed stromal cells occurred in response to Bcl-2 q indicating the effect is specific for malignant cells. Confocal and flow cytometry analysis of cells treated with FITC-labeled Bcl-2 q or MutBcl-2 showed prominent uptake and nuclear localization of Bcl-2 q in contrast to MutBcl-2. Inhibition of cell proliferation corresponded with decreased Bcl-2 protein expression and increased expression of pro-apoptotic proteins Bax and Bak. This resulted in decreased mitochondrial transmembrane potential and significant autophagic degradation of mitochondria, characteristic of mitophagy. Induction of mitophagy was associated with increased Beclin-1 and LC3B expression and disruption of Bcl-2/Beclin-1 interaction. These results demonstrate striking growth inhibition in response to Bcl-2 q related to inhibition of Bcl-2 expression and induction of mitochondrial autophagy and shows considerable therapeutic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3. doi:10.1158/1538-7445.AM2011-3

Abstract 2607: G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Many cancer-related pro-oncogenes have quadruplex forming oligonucleotide sequences in their promoter regions. These genes include, amongst others, c-Myc, Bcl-2, K-ras, or HIF-1a which are individually or together over-expressed in the vast majority of neoplasms. For instance, c-Myc, a key regulator of cell cycle and proliferation, is commonly over-expressed in both hematological and solid cancers. The c-myc promoter region contains a guanine-rich sequence (PU27), capable of forming four-stranded quadruplex DNA structures, which negatively regulates c-myc transcription. Also, Bcl-2, is commonly over-expressed in a variety of hematological malignancies and the Bcl-2 quadruplex-forming sequence is located upstream of the P1 promoter of the Bcl-2 gene; it is also implicated in negative regulation of Bcl-2 transcription. Treatment of cancer cells over-expressing c-Myc and / or Bcl-2 with oligonucleotides encoding the c-Myc (PU27) and Bcl-2 (Bcl-2-GRO) guanine-rich sequences, located in their respective promoter regions, selectively inhibits c-Myc or Bcl-2 gene expressions, cancer cells proliferation and induces cell death. Methods: Four cancer cell lines obtained from ATCC were used in these studies (Raji, HL-60, U937, A549). Cell Titer-Glo© Assay was used for the cell proliferation experiments. Cell cycle analysis was determined in the HL-60 cell line using fixed doses of PU27 and Bcl-2-GRO and time dependent FACS analysis. Internalization studies were analyzed by FACS analysis using FAM labeled PU27 and DY647 labeled Bcl-2-GRO. In vivo tumor growth inhibition studies were done in females NOD/SCID mice (6 animals per group); 5x106 cells in a Matrigel mixture were implanted in the flank of the animals; tumors developed and treatments with PU27 and Bcl-2-GRO at different dose levels alone and in combination were performed while body weights were monitored during the course of the study. Results: Treating the Raji, HL-60, U937, or A549 cancer cell lines with PU27 or Bcl-2-GRO causes inhibition of proliferation with IC50 between 200 nM and 5mM after 144 hours depending on expression of the target pro-oncogene. When treating the same cell lines using a combination of these two sequences, a significant synergy is observed, leading to more than 100 fold increase in potency. The same synergistic effect was seen when PU27 was combined with paclitaxel, leading to picomolar IC50. Internalization of these oligonucleotides was very rapid and quantitative. Localization studies show that PU27 and Bcl-2-GRO are localized in the nucleus. Cell cycle results showed a significantly increase in G0-G1 cell cycle arrest for the combination. In vivo xenograft tumor growth inhibition studies confirmed the in vitro synergistic results with 60 to 80% tumor growth inhibition while treatments were well tolerated (no body weight loss). Citation Format: Gilles H. Tapolsky, Kara Sedoris, Shelia Thomas, Francine Rezzoug, Donald M. Miller. G-rich DNA genomic sequences derived from the promoter region of pro-oncogenes selectively inhibit tumor growth and demonstrate strong synergies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2014-2607

Using Human Observations to Gain Biologic Insights and New Treatments; Discovery of a Quadruplex-Forming DNA Aptamer as an Anticancer Agent

The discovery and characterization of the primo vascular system represents a major new development in our understanding of human biology. The current understanding of this novel system is based on careful observations over an extended period of time. We have, in an analogous fashion, used clinical observations in patients with malignant diseases to develop new insights which have led to novel types of therapies. Beginning with the observation, in 1979, of in vivo differentiation of chronic myelogenous leukemia cells in a patient treated with the RNA synthesis inhibitor, mithramycin, we have characterized the ability of DNA binding compounds to inhibit gene expression. As a result of this work, we have discovered a quadruplex-forming, DNA aptamer, AS1411, which has significant anticancer activity with very little toxicity. AS1411 is currently in Phase IIb studies. We have recently shown that genomic quadruplex-forming sequences are selectively toxic to transformed cells and have therapeutic potential.

Abstract 4996: The VEGF quadruplex-forming sequence inhibits lung cancer cell growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGF q), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with an oligonucleotide encoding VEGF q inhibits cell growth by decreasing VEGF expression and subsequent signaling through VEGF receptor 2 (FLK-1). To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGF q or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Our results demonstrate that VEGF q formed a parallel quadruplex structure and showed remarkable serum and intracellular stability. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGF q showed prominent uptake and nuclear/cytoplasmic localization in contrast to MutVEGF. Treatment of A549 cells with VEGF q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay, which was not a result of cell cycle arrest. Matrigel analysis also showed a significant reduction in tumor invasiveness after 24 h with VEGF q treatment. Although uptake of VEGF q occurred in non-transformed Hs27 human fibroblast cells, no change in cell growth was evident, indicating that the anti-proliferative effects of VEGF q are specific for malignant cells. Inhibition of cell proliferation and invasion corresponded with decreased VEGF protein expression resulting in decreased FLK-1 signaling and phosphorylation of the target proteins ERK 1/2 and AKT. These results demonstrate striking growth inhibition and decreased invasion of A549 cells in response to VEGF q related to inhibition of VEGF expression and signaling through FLK-1 and have significant therapeutic implications for the treatment of non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4996. doi:10.1158/1538-7445.AM2011-4996

Abstract 2949: The VEGF quadruplex-forming sequence inhibits cell growth of lung cancer

Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGFq), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with VEGFq induces cell death by inhibiting gene expression. To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGFq or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of VEGF protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that VEGFq formed a stable parallel quadruplex structure, while the MutVEGF sequence did not form a quadruplex. Treatment of A549 cells with VEGFq caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2949.