Amanda R. Wasylishen

Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function

Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with beta1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA-induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC(50) of 60 +/- 20 nmol/L. Digoxin also increased the fraction of some concanavalin A-binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients.

Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

Myc The Beauty and the Beast

The iconic history of the Myc oncoprotein encompasses 3 decades of intense scientific discovery. There is no question that Myc has been a pioneer, advancing insight into the molecular basis of cancer as well as functioning as a critical control center for several diverse biological processes and regulatory mechanisms. This narrative chronicles the journey and milestones that have defined the understanding of Myc, and it provides an opportunity to consider future directions in this challenging yet rewarding field.

New model systems provide insights into Myc-induced transformation

The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure–function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (ΔMBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent.

MYC Phosphorylation at Novel Regulatory Regions Suppresses Transforming Activity

Despite its central role in human cancer, MYC deregulation is insufficient by itself to transform cells. Because inherent mechanisms of neoplastic control prevent precancerous lesions from becoming fully malignant, identifying transforming alleles of MYC that bypass such controls may provide fundamental insights into tumorigenesis. To date, the only activated allele of MYC known is T58A, the study of which led to identification of the tumor suppressor FBXW7 and its regulator USP28 as a novel therapeutic target. In this study, we screened a panel of MYC phosphorylation mutants for their ability to promote anchorage-independent colony growth of human MCF10A mammary epithelial cells, identifying S71A/S81A and T343A/S344A/S347A/S348A as more potent oncogenic mutants compared with wild-type (WT) MYC. The increased cell-transforming activity of these mutants was confirmed in SH-EP neuroblastoma cells and in three-dimensional MCF10A acini. Mechanistic investigations initiated by a genome-wide mRNA expression analysis of MCF10A acini identified 158 genes regulated by the mutant MYC alleles, compared with only 112 genes regulated by both WT and mutant alleles. Transcriptional gain-of-function was a common feature of the mutant alleles, with many additional genes uniquely dysregulated by individual mutant. Our work identifies novel sites of negative regulation in MYC and thus new sites for its therapeutic attack.

MYC activity is negatively regulated by a C-terminal lysine cluster

The MYC oncogene is not only deregulated in cancer through abnormally high levels of expression, but also through oncogenic lesions in upstream signalling cascades. Modelling MYC deregulation using signalling mutants is a productive research strategy. For example, the MYC threonine-58 to alanine substitution mutant (T58A) within MYC-homology box 1 is more transforming than wild-type (WT) MYC, because of decreased apoptosis and increased protein stability. Understanding the regulatory mechanisms controlling T58 phosphorylation has led to new approaches for the development of MYC inhibitors. In this manuscript, we have extensively characterized a MYC signalling mutant in which six lysine residues near the highly conserved MYC homology box IV and basic region have been substituted to arginines (6KR). Previous literature suggests these lysines can undergo both ubiquitylation and acetylation. We show MYC 6KR is able to fully rescue the slow growth phenotype of HO15.19 MYC-null fibroblasts, and promote cell cycle entry of serum-starved MCF10A cells. Remarkably, 6KR increased anchorage-independent colony growth compared with WT MYC in both SH-EP and MCF10A cells. Moreover, it was also more potent in promoting xenograft tumour growth of Rat1A and SH-EP cells. Combined, our data identify this region and these six lysines as important residues for the negative regulation of MYC-induced transformation. Mechanistically, we demonstrate that, unlike T58A, the increased transformation is not a result of increased protein stability or a reduced capacity for 6KR to induce apoptosis. Through expression analysis and luciferase reporter assays, we show that 6KR has increased transcriptional activity compared with WT MYC. Combined, through a comprehensive evaluation across multiple cell types, we identify an important regulatory region within MYC. A better understanding of the full scope of signalling through these residues will provide further insights into the mechanisms contributing to MYC-induced tumorigenesis and may unveil novel therapeutic strategies to target Myc in cancer.

Identification of c-MYC SUMOylation by Mass Spectrometry

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation.

Characterization of the apoptotic response of human leukemia cells to organosulfur compounds

BackgroundNovel therapeutic agents that selectively induce tumor cell death are urgently needed in the clinical management of cancers. Such agents would constitute effective adjuvant approaches to traditional chemotherapy regimens. Organosulfur compounds (OSCs), such as diallyl disulfide, have demonstrated anti-proliferative effects on cancer cells. We have previously shown that synthesized relatives of dysoxysulfone, a natural OSC derived from the Fijian medicinal plant, Dysoxylum richi, possess tumor-specific antiproliferative effects and are thus promising lead candidates.MethodsBecause our structure-activity analyses showed that regions flanking the disulfide bond mediated specificity, we synthesized 18 novel OSCs by structural modification of the most promising dysoxysulfone derivatives. These compounds were tested for anti-proliferative and apoptotic activity in both normal and leukemic cells.ResultsSix OSCs exhibited tumor-specific killing, having no effect on normal bone marrow, and are thus candidates for future toxicity studies. We then employed mRNA expression profiling to characterize the mechanisms by which different OSCs induce apoptosis. Using Gene Ontology analysis we show that each OSC altered a unique set of pathways, and that these differences could be partially rationalized from a transcription factor binding site analysis. For example, five compounds altered genes with a large enrichment of p53 binding sites in their promoter regions (p < 0.0001).ConclusionsTaken together, these data establish OSCs derivatized from dysoxysulfone as a novel group of compounds for development as anti-cancer agents.

More than MAX: Discovering the Myc interactome.

Comment on: Agrawal P, et al. Cell Cycle 2010; 9:4908-4921.

Abstract C165: Breast cancer, statins, and 3-D cell culture.

Statins are widely used to lower serum cholesterol levels. They act by inhibiting hydroxymethylglutaryl coenzyme A reductase (HMGCR), the rate-limiting step in the mevalonate pathway. Recent work also shows that statins could be used as anticancer therapeutics, particularly in breast cancer. This study presents a preliminary rationale for the use of statins as a therapy in breast cancer. We characterized a panel of breast cancer cell lines for sensitivity to fluvastatin, using proliferation and cell-death assays. We also screened for differences in activity of the electron transport chain and glycolysis after fluvastatin treatment. We then began to expand on these results using 3D cell culture techniques to offer a more representative tumor model. The panel of breast cancer cell lines showed a range of sensitivity to fluvastatin, with MTT50 for 72 h treatment varying from 0.7 μM for MDA-MB231 cells to 162.8 μM in BT474 cells. Interestingly, triple negative cell lines were in the sensitive range. Differences in cell cycle populations were also observed, with a representative panel of sensitive cells showing an increased G1/G0 arrest and decrease in S-phase population when compared to insensitive cell lines. Delving deeper, mitochondrial respiration was decreased in the sensitive cell lines. We also observed greater changes in morphology in the sensitive cell lines than the insensitive when grown in 3D cell culture, potentially offering more relevance to our observations. These results confirm that there is a range of sensitivities to fluvastatin in breast cancer cell lines, allowing for further studies to determine the cause of these differences. The hints at metabolic differences could also lead to novel co-treatments with statins and greater therapeutic benefit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C165.