Abstract Breast cancer is the most common cancer affecting women, with about 1.4 million new cases diagnosed each year. Triple-negative breast cancer (TNBC) which is characterized by lack of expression of estrogen, progesterone and human epidermal growth factor receptors. Is the most aggressive group of tumors associated with a poor prognosis (15% of all mammary tumors). Previous studies have shown that specific G-rich genomic sequences in the promoters of multiple human genes can form G-quadruplex structures, resulting in decreased transcription and gene expression. These regions are generally found within gene promoters especially of oncogenes such as c-MYC, KRAS, VEGF, BCL2 or hTERT. Most of these genes are abnormally expressed in breast cancer. The c-MYC gene regulates a large array of genes essential for cell functions including proliferation, metabolism, differentiation, adhesion and apoptosis. We have recently shown that oligonucleotides encoding the G-quadruplex sequence of the c-MYC, VEGF or hTERT promoters down regulate expression of their respective genes and inhibit cell proliferation. We hypothesized that such oligonucleotides could be applied to breast cancer cell lines in order to inhibit cell proliferation and metastasis. In this study, we evaluated the effect of oligonucleotides targeted to c-MYC (Pu27 and Pu27 Palmi), KRAS (KRASq), VEGF (VEGFq), hTERT (Tert-FL) and BCL2 (BCL2q) in Breast cancer cell lines (MDA-MB-231, SKBR3, MCF7) and MCF10A (non-transformed mammary cells). The effect of each oligonucleotide on cell growth was evaluated using the MTT assay. Our results reveal that all oligos inhibit cell growth of MDA-MB-231. However, for SKBR3 and MCF7, only Tert-FL had growth inhibitory activity. The control cell line, MCF10A, did not respond suggesting that the oligonucleotides affect preferentially tumor cells. The effect of the oligonucleotides on gene expression (qRT-PCR) was performed on MDA-MB-231 exposed for 3 days. The analysis of gene expression shows the downregulation of c-MYC by Pu27 and hTERT by Tert-FL suggesting a direct effect on gene expression. In addition, Pu27 downregulated hTERT and VEGF which are both under c-MYC control. Since MDA-MB-231 is a TNBC cell line overexpressing c-MYC and is highly enriched in cancer stem cells, we evaluate the effect of Pu27 and Tert-FL on the tumorsphere formation. The results showed a very strong inhibition of tumorsphere formation in the cells treated with Pu27. Our findings suggest that oligonucleotides which target genes such as c-MYC, hTERT or KRAS very efficiently inhibit the growth of BC cell lines in particular MDA-MB-231 TN most likely by downregulating target gene expression. In addition, we demonstrate that downregulating c-MYC expression Pu27 dramatically reduces the cancer stem cell numbers. The use of G-quadruplex forming oligonucleotides targeted to c-MYC and /or hTERT may constitute a new therapeutic strategy especially for TNBC where targeted therapy is lacking. Citation Format: Elaine Stur, Shelia Thomas, Francine Rezzoug, Donald Miller. Down-regulation of c-MYC and hTERT gene expression in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1520. doi:10.1158/1538-7445.AM2017-1520
The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.
G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.
Abstract AS1411 is a quadruplex-forming oligonucleotide aptamer that targets nucleolin. It is currently in clinical trials as a treatment for various cancers. We have proposed that AS1411 inhibits cancer cell proliferation by affecting the activities of certain nucleolin-containing complexes. Here, we report that protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the formation of symmetrical dimethylarginine (sDMA), is a nucleolin-associated protein whose localization and activity are altered by AS1411. Levels of PRMT5 were found to be decreased in the nucleus of AS1411-treated DU145 human prostate cancer cells, but increased in the cytoplasm. These changes were dependent on nucleolin and were not observed in cells pretreated with nucleolin-specific small interfering RNA. Treatment with AS1411 altered levels of PRMT5 activity (assessed by sDMA levels) in accord with changes in its localization. In addition, our data indicate that nucleolin itself is a substrate for PRMT5 and that distribution of sDMA-modified nucleolin is altered by AS1411. Because histone arginine methylation by PRMT5 causes transcriptional repression, we also examined expression of selected PRMT5 target genes in AS1411-treated cells. For some genes, including cyclin E2 and tumor suppressor ST7, a significant up-regulation was noted, which corresponded with decreased PRMT5 association with the gene promoter. We conclude that nucleolin is a novel binding partner and substrate for PRMT5, and that AS1411 causes relocalization of the nucleolin-PRMT5 complex from the nucleus to the cytoplasm. Consequently, the nuclear activity of PRMT5 is decreased, leading to derepression of some PRMT5 target genes, which may contribute to the biological effects of AS1411.
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.
Abstract Throughout the genome as well as telomere region, there is presence of DNA sequences which forms quadruplex. Here we show Pu-27 triggers the damage of cells by interfering telomeric ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks (DSB). We found that Pu-27 frequently prompted chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal break (27%) by karyotyping of U937 metaphase cells and was associated with induction of DNA damage response sensor γ-H2AX. Analysis of DNA damage response regulators by RT-PCR array of Pu-27 treated U937 cells revealed downregulation of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), upstream kinase ATM, DNA damage response mediators (RAD17, RAD50 and 53BP1), and cell cycle arrest molecules (CHK1 and CHK2). Interestingly, there were no changes or low expression of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene TERT respectively. αB-U937 cell, where basic-domain of shelterin protein TRF2 was deleted became relatively resistance to Pu-27 and showed no changes of constitutively active form of γ-H2AX. RT-PCR array of Pu-27 treated αB-U937 cell appeared to be no changes of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), DNA damage response mediators (RAD17, RAD50 and 53BP1); down regulation of upstream kinase ATM; up regulation of cell cycle arrest molecules (SMC1, CHK1 and CHK2), DNA repair molecules (H2AX and BRCA1), and telomere maintenance gene TERT. Shelterin protein, TRF1 co-associated with γH2AX by inducing TIFs (Telomere-dysfunction Induced Foci) when U937 cell was treated with Pu-27. Sk-Lu-1, an Alt (Alternating Lengthening to Telomere) cell which does not use TERT for their growth and propagation is relatively resistance to Pu-27. ATM preserves the telomeric homeostasis by maintaining the integrity of ‘shelterin’ protein. To substantiate the specificity of Pu-27 to shelterin protein complex, we found ATM-deficient cell was more sensitive to Pu-27 than ATM-proficient cell. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these results suggest that Pu-27-treated sensitivity at least in part due to compromising telomeric shelterin complex and perhaps stabilizing telomeric quadruplex and not by directly inhibiting telomerase enzyme. U937 cell with chromosomal aberration and unstable telomere progress through cell cycle without arrest and repair leading to cell death whereas αB-U937 cells is continuously repaired by arresting and induction of repair machinery. Continuous exposure of Pu-27 results in refractory to repair of DNA damage leading to uncompensated genomic stability leading to cell death. Citation Format: Md Ashraful Islam, Shelia D. Thomas, Kara J. Sedoris, Donald M. Miller. Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2013-743 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Vascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression.Our experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death.Our data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression.
Alpha-enolase is a bifunctional gene encoding both a glycolytic enzyme and a DNA binding protein, c-myc binding protein (MBP-1). MBP-1 binds the c-myc promoter and downregulates c-myc transcription. Since these alpha-enolase gene products have important functions in glucose metabolism and growth regulation, this gene may play a central role in regulating the abnormal proliferative characteristics of transformed cells. To determine the role of alpha-enolase and MBP-1 in the cellular response to altered exogenous glucose concentration, MCF-7 cells were cultured in low (1 nM), physiological (5 mM), or high (25 mM) levels of glucose. Levels of alpha-enolase, MBP-1, and c-myc expression were compared to levels of cell proliferation and lactate production. At all glucose concentrations, MCF-7 cells demonstrated an initial increase in MBP-1 expression and a parallel decrease in c-myc transcript levels, which were accompanied by decreased proliferation. Cells grown in low glucose maintained the increased MBP-1 expression through 48 h, resulting in persistently lower rates of proliferation. However, physiologic or high glucose levels resulted in decreased MBP-1 expression, which was associated with increased cellular proliferation and lactate production. In these cells, c-myc mRNA returned to control levels as MBP-1 expression decreased. Cells grown in low glucose demonstrated a dramatic increase in c-myc mRNA at 48 h, which was associated with a loss in c-myc P2 promoter binding by MBP-1. This suggests that post-translational modifications of MBP-1 likely alter its DNA binding activity. These results demonstrate an important role for MBP-1 in the altered cell proliferation and energy utilization that occur in response to an altered glucose concentration.
Neuroblastoma is the most common solid tumor of childhood malignancy. The biological properties vary from indolent to aggressive depending on how different genes play their role in transforming neural crest cells leading to spontaneous regression to an unfavorable outcome. Guanine-Rich Oligonucleotides (GRO) are being tested as a potential target for an anti-cancer drug. In this study, we have identified GROs at the promoter regions of the MYCN oncogene (MYCN-15) and it formed quadruplex in-vitro shown by circular dichroism (CD). Then three neuroblastoma cell lines were used to explore the effect of MYCN-15 on different biological properties: cellular growth, differentiation, and death. The neuroblastoma cell lines SH-SY5Y and SK-N-AS have a single copy of the MYCN gene, whereas the SK-N-BE2 cell line has multiple copies (n-Myc amplified). MYCN-15-induced cellular differentiation in both SK-N-AS and SH-SY5Y cells was followed by cell death in the SH-SY5Y cell line only. MTT [3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide] assay revealed, that there was modest growth inhibition in both SH-SY5Y (~58%) and SK-N-BE2 (~38%) cells when treated with MYCN-15 oligonucleotide. There were no significant changes in survivin protein expression in both SK-N-AS and SH-SY5Y cells when treated with MYCN-15 oligonucleotides. Taken together, MYCN-15 can be used as an essential target oligonucleotide for treating human neuroblastoma.
