N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification.     

Combined RAR alpha- and RXR-specific ligands overcome N-myc-associated retinoid resistance in neuroblastoma cells

Retinoids induce human neuroblastoma cells to undergo growth inhibition and neuritic differentiation in vitro, through interactions with nuclear retinoid receptor proteins. In this study, we found that three different neuroblastoma cell lines exhibited wide variation in their responsiveness to the growth inhibitory effects of the retinoic acid receptor (RAR) agonist, all-trans-retinoic acid (aRA). Resistance to the growth inhibitory effect of aRA correlated with the presence of N-myc gene amplification and not aRA-induced RAR beta levels. Over-expression of N-myc in a neuroblastoma cell line with no endogenous N-myc expression caused a marked reduction in retinoid-induced growth inhibition. Combination of receptor-specific retinoid agonists for RXR and RAR alpha significantly enhanced the sensitivity of N-myc-amplified neuroblastoma cells to the growth inhibitory effects of aRA. Our results indicate that combination receptor-specific retinoid therapy can overcome N-myc-mediated retinoid resistance and may be a more effective chemo-preventive strategy in the disease.     

The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells.

Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.     

Upregulation of SATB1 Is Associated with Prostate Cancer Aggressiveness and Disease Progression

Disease aggressiveness remains a critical factor to the progression of prostate cancer. Transformation of epithelial cells to mesenchymal lineage, associated with the loss of E-cadherin, offers significant invasive potential and migration capability. Recently, Special AT-rich binding protein (SATB1) has been linked to tumor progression. SATB1 is a cell-type restricted nuclear protein, which functions as a tissue-specific organizer of DNA sequences during cellular differentiation. Our results demonstrate that SATB1 plays significant role in prostate tumor invasion and migration and its nuclear localization correlates with disease aggressiveness. Clinical specimen analysis showed that SATB1 was predominantly expressed in the nucleus of high-grade tumors compared to low-grade tumor and benign tissue. A progressive increase in the nuclear levels of SATB1 was observed in cancer tissues compared to benign specimens. Similarly, SATB1 protein levels were higher in a number of prostate cancer cells viz. HPV-CA-10, DU145, DUPro, PC-3, PC-3M, LNCaP and C4-2B, compared to non-tumorigenic PZ-HPV-7 cells. Nuclear expression of SATB1 was higher in biologically aggressive subclones of prostate cancer cells with their respective parental cell lines. Furthermore, ectopic SATB1 transfection conferred increased cell motility and invasiveness in immortalized human prostate epithelial PZ-HPV-7 cells which correlated with the loss of E-cadherin expression. Consequently, knockdown of SATB1 in highly aggressive human prostate cancer PC-3M cells inhibited invasiveness and tumor growth in vivo along with increase in E-cadherin protein expression. Our findings demonstrate that SATB1 has ability to promote prostate cancer aggressiveness through epithelial-mesenchymal transition.     

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.     

N-Myc Regulates Expression of Pluripotency Genes in Neuroblastoma Including lif,klf2, klf4, and lin28b

myc genes are best known for causing tumors when overexpressed, but recent studies suggest endogenous myc regulates pluripotency and self-renewal of stem cells. For example, N-myc is associated with a number of tumors including neuroblastoma, but also plays a central role in the function of normal neural stem and precursor cells (NSC). Both c- and N-myc also enhance the production of induced pluripotent stem cells (iPSC) and are linked to neural tumor stem cells. The mechanisms by which myc regulates normal and neoplastic stem-related functions remain largely open questions. Here from a global, unbiased search for N-Myc bound genes using ChIP-chip assays in neuroblastoma, we found lif as a putative N-Myc bound gene with a number of strong N-Myc binding peaks in the promoter region enriched for E-boxes. Amongst putative N-Myc target genes in expression microarray studies in neuroblastoma we also found lif and three additional important embryonic stem cell (ESC)-related factors that are linked to production of iPSC: klf2, klf4, and lin28b. To examine the regulation of these genes by N-Myc, we measured their expression using neuroblastoma cells that contain a Tet-regulatable N-myc transgene (TET21N) as well as NSC with a nestin-cre driven N-myc knockout. N-myc levels closely correlated with the expression of all of these genes in neuroblastoma and all but lif in NSC. Direct ChIP assays also indicate that N-Myc directly binds the lif promoter. N-Myc regulates trimethylation of lysine 4 of histone H3 in the promoter of lif and possibly in the promoters of several other stem-related genes. Together these findings indicate that N-Myc regulates overlapping stem-related gene expression programs in neuroblastoma and NSC, supporting a novel model by which amplification of the N-myc gene may drive formation of neuroblastoma. They also suggest mechanisms by which Myc proteins more generally contribute to maintenance of pluripotency and self-renewal of ESC as well as to iPSC formation.     

Src Kinase Regulation in Progressively Invasive Cancer

Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.     

ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.     

Polyamine Metabolism Is Sensitive to Glycolysis Inhibition in Human Neuroblastoma Cells

Polyamines are essential for cell proliferation, and their levels are elevated in many human tumors. The oncogene n-myc is known to potentiate polyamine metabolism. Neuroblastoma, the most frequent extracranial solid tumor in children, harbors the amplification of n-myc oncogene in 25% of the cases, and it is associated with treatment failure and poor prognosis. We evaluated several metabolic features of the human neuroblastoma cell lines Kelly, IMR-32, and SK-N-SH. We further investigated the effects of glycolysis impairment in polyamine metabolism in these cell lines. A previously unknown linkage between glycolysis impairment and polyamine reduction is unveiled. We show that glycolysis inhibition is able to trigger signaling events leading to the reduction of N-Myc protein levels and a subsequent decrease of both ornithine decarboxylase expression and polyamine levels, accompanied by cell cycle blockade preceding cell death. New anti-tumor strategies could take advantage of the direct relationship between glucose deprivation and polyamine metabolism impairment, leading to cell death, and its apparent dependence on n-myc. Combined therapies targeting glucose metabolism and polyamine synthesis could be effective in the treatment of n-myc-expressing tumors.     

Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma

High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells

Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.