Stewart A. Smith

Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis

Histone deacetylase (HDAC) inhibitors reactivate tumor suppressor gene transcription; induce cancer cell differentiation, growth arrest, and programmed cell death; and are among the most promising new classes of anticancer drugs. Myc oncoproteins can block cell differentiation and promote cell proliferation and malignant transformation, in some cases by modulating target gene transcription. Here, we show that tissue transglutaminase (TG2) was commonly reactivated by HDAC inhibitors in neuroblastoma and breast cancer cells but not normal cells and contributed to HDAC inhibitor-induced growth arrest. TG2 was the gene most significantly repressed by N-Myc in neuroblastoma cells in a cDNA microarray analysis and was commonly repressed by N-Myc in neuroblastoma cells and c-Myc in breast cancer cells. Repression of TG2 expression by N-Myc in neuroblastoma cells was necessary for the inhibitory effect of N-Myc on neuroblastoma cell differentiation. Dual step cross-linking chromatin immunoprecipitation and protein coimmunoprecipitation assays showed that N-Myc acted as a transrepressor by recruiting the HDAC1 protein to an Sp1-binding site in the TG2 core promoter in a manner distinct from its action as a transactivator at E-Box binding sites. HDAC inhibitor treatment blocked the N-Myc-mediated HDAC1 recruitment and TG2 repression in vitro. In neuroblastoma-bearing N-Myc transgenic mice, HDAC inhibitor treatment induced TG2 expression and demonstrated marked antitumor activity in vivo. Taken together, our data indicate the critical roles of HDAC1 and TG2 in Myc-induced oncogenesis and have significant implications for the use of HDAC inhibitor therapy in Myc-driven oncogenesis.

Favorable prognostic significance of high-level retinoic acid receptor β expression in neuroblastoma mediated by effects on cell cycle regulation

We have previously shown that ectopic overexpression of retinoic acid receptor (RAR) subtypes α, β and γ in human neuroblastoma cells had different effects on growth and retinoid sensitivity. Only overexpressed RARβ induced profound growth inhibition in the absence of additional retinoid, and increased retinoid sensitivity. In this study, we measured mRNA expression levels of RARα, β, and γ in 50 primary neuroblastoma tumor samples, and found a strong correlation between favorable patient prognosis and high-level RARβ expression. Human neuroblastoma cells transfected with a vector expressing RARβ demonstrated irreversible growth arrest following a 1 week exposure to all-trans-retinoic acid, whereas control cells continued to proliferate. In the absence of additional retinoid, RARβ transfectants demonstrated a higher proportion of cells in the G0/G1 phase of the cell cycle, increased p21WAF1/CIP1 expression and specific binding to a retinoic acid response element. These were changes which we also observed in control neuroblastoma cells following retinoid treatment. Our data indicate that RARβ is an important factor mediating the growth inhibitory effects of retinoids in neuroblastoma cells. The favorable effect of high-level RARβ expression on prognosis in primary tumor tissue may occur through RARβ effects on p21 expression and consequent G0/G1 cell cycle arrest.

The retinoid anticancer signal: mechanisms of target gene regulation

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARβ2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARβ2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARβ2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARβ2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARβ2, DUSP6, and RGS16.

Combined RARα- 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 Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal *

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor β (RARβ) gene transcription. The RA response element β (βRARE) is the essential DNA sequence required for retinoid-induced RARβ transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a βRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARβ transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARβ expression. EBBP increased βRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism.

Growth inhibitory retinoid effects after recruitment of retinoid X receptor β to the retinoic acid receptor β promoter

Nuclear retinoid receptors mediate retinoid effects through tissue‐specific, ligand–receptor interactions and subsequent transcriptional regulation of secondary target genes. Retinoic acid receptor beta (RARβ) is itself a retinoid target gene with a retinoic acid response element (βRARE) in the 5′ untranslated region of the RARβ2 gene. Altered transcriptional regulation of RARβ may play a role in human carcinogenesis and the retinoid‐responsiveness of malignant cells. Here we used retinoid X receptor‐specific antibodies in electrophoretic mobility shift assays to show that the retinoid X receptor β (RXRβ) protein was recruited to the βRARE, after retinoid treatment of retinoid‐sensitive neuroblastoma (NB), lung and breast cancer cell lines, but not retinoid‐resistant lung and breast cancer cell lines. RXRβ selectively enhanced retinoid‐induced transcriptional activation of the βRARE. Stable overexpression of RXRα and RXRβ in NB cells resulted in marked growth inhibition and cell death, which increased after retinoid treatment. However, only proteins from the RXRβ transfectants exhibited specific RXRβ binding to the βRARE in vitro and in vivo, enhanced histone acetylation and increased endogenous RARβ expression. These data indicate that recruitment of RXRβ to the βRARE, and consequent induction of endogenous RARβ expression, is an important component in the retinoid anticancer signal. RXRα may also participate in the retinoid signal, but through mechanisms that do not involve RARβ.

Cloning and characterization of the human neural cell adhesion molecule, CNTN4 (alias BIG-2)

We report the isolation and characterization of human contactin 4 (CNTN4), a brain-derived, immunoglobulin superfamily molecule-2 (alias BIG-2) as a candidate gene responsible for the differentiation potential of human neuroblastoma cells. Northern blot analysis showed highest CNTN4 expression in testes, thyroid, small intestine, uterus and brain. Induction of CNTN4 mRNA expression in human neuroblastoma tumor cells treated with retinoic acid correlated with a block in retinoid-induced neuritogenesis. Our findings suggest a role for human contactin 4 protein in the response of neuroblastoma cells to differentiating agents.

Expression of the putative tumour suppressor gene, p73, in neuroblastoma and other childhood tumours.

BACKGROUND The recently characterised p53 homologue, p73, has been mapped to chromosome 1p36, a region often found deleted in neuroblastoma. Although p73 has been implicated as a candidate tumour suppressor for neuroblastoma, mutations in the gene appear to be rare, thus suggesting other mechanisms for its aberrant behaviour. PROCEDURE We analysed p73 gene expression in a panel of primary neuroblastoma tumours and cell lines, as well as other tumours of childhood, using a reverse transcriptase-polymerase chain reaction assay. RESULTS Although low-level p73 expression was detected in 44/45 primary neuroblastoma tumours, no association with prognostic markers could be discerned. In no case was the level of p73 expression in the primary tumours as high as that observed in p73-expressing neuroblastoma cell lines. Expression of the p73 gene was also detected in 24/34 other childhood tumours. CONCLUSION Collectively, the data raise doubts over the role of this gene as a tumour suppressor in neuroblastoma.