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Supplementary Data from Dysregulation of Platelet-Derived Growth Factor β-Receptor Expression by ΔNp73 in Neuroblastoma

We have previously characterized how p53 family proteins control the transcriptional regulation of the platelet-derived growth factor β-receptor (PDGFRB) and found that ΔNp73α, acting dominant-negatively to p53 and p73, can upregulate PDGFRB promoter activity. Here, we report that PDGFRB regulation differs between two neuroblastoma cell lines, correlating with the actions of ΔNp73. We found that PDGFRB was highly expressed in IMR-32 cells, and serum stimulation of IMR-32 cells did not downregulate PDGFRB expression, as seen in SH-SY5Y cells. In IMR-32, ΔNp73 was found constitutively bound to the PDGFRB promoter, and silencing of ΔNp73 resulted in repression of PDGFRB promoter activity as well as decreased PDGFRB protein expression. However, the anticancer drug cisplatin, known to stabilize and activate p53 and p73, downregulated PDGFRB expression not only in SH-SY5Y but also in IMR-32. Chromatin immunoprecipitation showed that cisplatin removed ΔNp73 from the PDGFRB promoter and recruited p53 and p73, leading to binding of histone deacetylase 4. These results suggest a direct role of ΔNp73 in the constantly enhanced PDGFRB expression seen in tumors. (Mol Cancer Res 2009;7(12):2031–9)

Purpose: Our previous study showed that expression of NLRR3 is significantly high in favorable neuroblastomas (NBL), whereas that of NLRR1 is significantly high in unfavorable NBLs. However, the molecular mechanism of transcriptional regulation of NLRR3 remains elusive. This study was undertaken to clarify the transcriptional regulation of NLRR3 and its association with the prognosis of NBL.Experimental Design:NLRR3 and MYCN expressions in NBL cell lines were analyzed after induction of cell differentiation, MYCN knockdown, and overexpression. The transcriptional regulation of NLRR3 was analyzed by luciferase reporter and chromatin immunoprecipitation assays. Quantitative PCR was used for examining the expression of NLRR3, Miz-1, or MYCN in 87 primary NBLs.Results: The expression of NLRR3 mRNA was upregulated during differentiation of NBL cells induced by retinoic acid, accompanied with reduced expression of MYCN, suggesting that NLRR3 expression was inversely correlated with MYCN in differentiation. Indeed, knockdown of MYCN induced NLRR3 expression, whereas exogenously expressed MYCN reduced cellular NLRR3 expression. We found that Miz-1 was highly expressed in favorable NBLs and NLRR3 was induced by Miz-1 expression in NBL cells. MYCN and Miz-1 complexes bound to NLRR3 promoter and showed a negative regulation of NLRR3 expression. In addition, a combination of low expression of NLRR3 and high expression of MYCN was highly associated with poor prognosis.Conclusions:NLRR3 is a direct target of MYCN, which associates with Miz-1 and negatively regulates NLRR3 expression. NLRR3 may play a role in NBL differentiation and the survival of NBL patients by inversely correlating with MYCN amplification. Clin Cancer Res; 17(21); 6681–92. ©2011 AACR.

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During the early phase of tumorigenesis, primary malignant cells survive within a low nutrition environment caused by a poorly organized vascular system. Here, we sought to determine the functional significance of CD133 in the survival of cancer cells under nutrient‐poor conditions. Knockdown and overexpression experiments demonstrated that CD133 suppresses colon cancer cell death induced by serum deprivation through activation of Akt‐mediated anti‐apoptosis and protein synthesis pathways. Furthermore, serum deprivation increased the amount of endogenous CD133 protein, which was regulated at least in part by phosphoinositide 3‐kinase. Thus, it is highly likely that CD133 contributes to the acquisition/maintenance of the resistance to stress arising from nutrient deficiency in early avascular tumor tissues., During tumorigenesis, primary malignant cells survive within a low nutrition environment as a result of a poorly organized vascular system. Here, we investigated the significance of CD133 in the survival of cancer cells under nutrient‐poor conditions. Our results suggest that CD133 contributes to the growth of tumors, in which nutrients are poorly supplied, through Akt‐mediated anti‐apoptosis and protein synthesis pathways.

TAp63 is an isoform of p63 gene, a p53 family gene that suppresses tumorigenesis via transcriptional regulation. TAp63 represses transcription of MYC oncogene in glioblastomas; however, its role in another MYC family gene, MYCN, has remained elusive. In this study, we showed that TAp63 repressed transcription of the MYCN gene in human cancer cells. Overexpression of TAp63 in HeLa cells suppressed MYCN expression, whereas knockdown of TAp63 had the opposite effect. By binding to exon 1 of MYCN gene, TAp63 suppressed the promoter activities of MYCN and its cis-antisense gene, NCYM. Other p53 family members, p53 and TAp73, showed lesser ability to suppress MYCN/NCYM promoter activities compared with that of TAp63. All-trans-retinoic acid (ATRA) treatment of MYCN/NCYM-amplified neuroblastoma CHP134 cells induced TAp63 and reduced p53 expressions, accompanied by downregulation of MYCN/NCYM expressions. Meanwhile, TAp63 knockdown inhibited ATRA-induced repression of NCYM gene expression. Blocking the p53 family binding sites by CRISPR-dCas9 system in CHP134 cells induced MYCN/NCYM expression and promoted apoptotic cell death. Expression levels of TAp63 mRNA inversely correlated with those of MYCN/NCYM expression in primary neuroblastomas, which was associated with a favorable prognosis. Collectively, TAp63 repressed MYCN/NCYM bidirectional transcription, contributing to the suppression of neuroblastoma growth.

Receptor-type protein tyrosine phosphatase κ (PTPRK) is considered to be a candidate tumor suppressor. PTPRK dephosphorylates CD133, which is a stem cell marker; phosphorylated CD133 accelerates xenograft tumor growth of colon cancer cells through the activation of AKT, but the functional significance of this has remained elusive. In this study, we have demonstrated that knockdown of PTPRK potentiates the pro-oncogenic CD133-AKT pathway in colon cancer cells. Intriguingly, depletion of PTPRK significantly reduced sensitivity to the anti-cancer drug oxaliplatin and was accompanied by up-regulation of phosphorylation of Bad, a downstream target of AKT. Together, our present observations strongly suggest that the CD133-PTPRK axis plays a pivotal role in the regulation of colon cancer progression as well as drug resistance.

KIF1Bβ, a member of the kinesin superfamily of motor proteins, is a haploinsufficient tumor suppressor mapped to chromosome 1p36.2, which is frequently deleted in neural crest–derived tumors, including neuroblastoma and pheochromocytoma. While KIF1Bβ acts downstream of the nerve growth factor (NGF) pathway to induce apoptosis, further molecular functions of this gene product have largely been unexplored. In this study, we report that KIF1Bβ destabilizes the morphological structure of mitochondria, which is critical for cell survival and apoptosis. We identified YME1L1, a mitochondrial metalloprotease responsible for the cleavage of the mitochondrial GTPase OPA1, as a physical interacting partner of KIF1Bβ. KIF1Bβ interacted with YME1L1 through its death‐inducing region, as initiated the protease activity of YME1L1 to cleave the long forms of OPA1, resulting in mitochondrial fragmentation. Overexpression of YME1L1 promoted apoptosis, while knockdown of YME1L1 promoted cell growth. High YME1L1 expression was significantly associated with a better prognosis in neuroblastoma. Furthermore, in NGF‐deprived PC12 cells, KIF1Bβ and YME1L1 were upregulated, accompanied by mitochondrial fragmentation and apoptotic cell death. Small interfering RNA–mediated knockdown of either protein alone, however, remarkably inhibited the NGF depletion–induced apoptosis. Our findings indicate that tumor suppressor KIF1Bβ plays an important role in intrinsic mitochondria–mediated apoptosis through the regulation of structural and functional dynamics of mitochondria in collaboration with YME1L1. Dysfunction of the KIF1Bβ/YME1L1/OPA1 mechanism may be involved in malignant biological features of neural crest–derived tumors as well as the initiation and progression of neurodegenerative diseases.

Tumor suppressor p53 is a short‐lived nuclear transcription factor, which becomes stabilized and activated in response to a wide variety of cellular stresses. Around 50% of human cancer tissues carry p53 mutations, and certain p53 mutations contribute to chemoresistance. In the present study, we found that histone deacetylase 2 (HDAC2) acts as a co‐activator of tumor suppressor p53 and participates in the early molecular events following DNA damage. Anti‐cancer drug adriamycin (ADR) treatment induced cell death in p53‐wild‐type human osteosarcoma U2OS cells, and this was accompanied by a remarkable accumulation of p53 and γH2AX. HDAC2 gene silencing significantly decreased the sensitivity of U2OS cells to ADR and attenuated p53‐dependent DNA damage responses, such as ADR‐mediated phosphorylation of ataxia telangiectasia mutated (ATM) and p53, as well as accumulation of γH2AX and cleaved poly (ADP‐ribose) polymerase. However, HDAC2 knockdown had a marginal effect on p53‐null human lung cancer H1299 cells following ADR exposure. In contrast, forced expression of HA‐HDAC2 promoted cell death and stimulated the transcriptional activity of p53. Moreover, p53 and HDAC2 were found to co‐precipitate with ATM. Together, our present results strongly suggest that the p53–HDAC2 axis plays a vital role in the regulation of the DNA damage response and also contributes to chemosensitivity of cancer cells.

Since human bladder cancer (BC) is a common malignancy of the urinary system with poor prognosis, it is crucial to clarify the molecular mechanisms of BC development and progression. To the best of our knowledge, the current study demonstrated for the first time that miR-489-3p suppressed BC cell-derived tumor growth in vivo via the downregulation of histone deacetylase 2 (HDAC2). According to the results, expression levels of miR-489-3p were lower in BC tissues compared with corresponding normal tissues. Expression of miR-489-3p mimics in BC-derived T24 and 5637 cells resulted in a significant reduction in proliferation and migration rates. Furthermore, bioinformatics analyses indicated that HDAC2 may be a potential downstream target of miR-489-3p. In contrast to miR-489-3p, HDAC2 was expressed at higher levels in BC tissues compared with corresponding normal tissues. Additionally, small interfering RNA-mediated knockdown of HDAC2 caused a marked decrease in the proliferation and migration rates of T24 and 5637 cells. Consistent with these observations, expression of miR-489-3p mimics attenuated the growth of xenograft tumors arising from T24 cells and resulted in HDAC2 downregulation. In conclusion, the results of the current study indicated that the miR-489-3p/HDAC2 axis serves a role in the development and/or the progression of BC and may be a potential molecular target for the development of a novel strategy to treat patients with BC.

Background Chemo-resistance of bladder cancer has been considered to be one of the serious issues to be solved. In this study, we revealed pivotal role of miR-424 in the regulation of CDDP sensitivity of bladder cancer cells. Methods The cytotoxicity of cisplatin and effect of miR-424 were assessed by flow cytometry and TUNEL. Transcriptional regulation of miR-424 by HIF-1α was assessed by Chromatin immunoprecipitation (ChIP). Effect of miR-424 on expression of UNC5B, SIRT4 (Sirtuin4) and apoptotic markers was measured by QRT-PCR and/or Western blot. The regulation of miR-424 for UNC5B and SIRT4 were tested by luciferase reporter assay. The 5637-inoculated nude mice xenograft model was used for the in vivo study. The clinical significance of miR-424 was demonstrated mainly through data mining and statistical analysis of TCGA. Results In this study, we have found for the first time that cisplatin (CDDP) induces the expression of miR-424 in a HIF-1α-dependent manner under normoxia, and miR-424 plays a vital role in the regulation of CDDP resistance of bladder cancer cells in vitro. Mechanistically, we have found that UNC5B and SIRT4 are the direct downstream target genes of miR-424. CDDP-mediated suppression of xenograft bladder tumor growth was prohibited by the addition of miR-424, whereas ectopic expression of UNC5B or SIRT4 partially restored miR-424-dependent decrease in CDDP sensitivity of bladder cancer 5637 and T24 cells. Moreover, knockdown of UNC5B or SIRT4 prohibited CDDP-mediated proteolytic cleavage of PARP and also decreased CDDP sensitivity of these cells. Consistently, the higher expression levels of miR-424 were closely associated with the poor clinical outcome of the bladder cancer patients. There existed a clear inverse relationship between the expression levels of miR-424 and pro-apoptotic UNC5B or SIRT4 in bladder cancer tissues. Conclusions Collectively, our current results strongly suggest that miR-424 tightly participates in the acquisition/maintenance of CDDP-resistant phenotype of bladder cancer cells through down-regulation of its targets UNC5B and SIRT4, and thus combination chemotherapy of CDDP plus HIF-1α/miR-424 inhibition might have a significant impact on hypoxic as well as normoxic bladder cancer cells.

E2F transcription factor 5 (E2F5) is a member of the E2F family of transcription factors, which are involved in regulation of various cellular processes, including cellular proliferation, apoptosis, differentiation and DNA damage response. Previously, we reported that E2F5 was aberrantly overexpressed in estrogen receptor (ER)‑negative breast cancer, especially in triple‑negative breast cancer (TNBC). In the present study, it was revealed that E2F5 gene silencing caused a significant reduction in the proliferation rate of breast cancer MCF7 (ER‑positive luminal‑type) and MDA‑MB‑231 (TNBC‑type) cells. Additional experiments demonstrated that E2F5 knockdown triggered cell death of MCF7 cells but not MDA‑MB‑231 cells. As MCF7 and MDA‑MB‑231 cells carry wild‑type and mutant TP53, respectively, and BT474 (ER‑negative, HER2‑positive type) carrying mutant TP53 exhibited similar results to MDA‑MB‑231, the possible effects of E2F5 gene depletion on cell death‑related TP53‑target gene expression were examined. Real‑time RT‑qPCR analysis revealed that knockdown of E2F5 in MCF7 cells stimulated cell death‑related transcription of TP53‑target genes such as BAX, NOXA and PUMA. For MDA‑MB‑231 and BT474 cells, E2F5 gene silencing revealed marginal effects on the expression of TP53 target genes. In addition, silencing of TP53 abrogated the effect of E2F5 silencing in MCF7 cells. Collectively, the present results indicated that E2F5 participated in the carcinogenesis of breast cancer carrying wild‑type TP53 through suppression of TP53, while E2F5 had a pro‑proliferative but not anti‑apoptotic effect on breast cancer with TP53 mutation.

High in-field performance and low anisotropy of critical current density J c are important for superconducting wires/tapes in rotating machine and magnet applications. Here, we report measurements of angular dependence of critical current density J c in proton-irradiated FeSe 0.5 Te 0.5 (FST) films at magnetic field up to 35 T. We observed two-fold reduction in the J c anisotropy of FST films at 4.2 K and 15 Tesla magnetic field by irradiation with low-energy (190 keV) proton. In light of recent demonstration of roll-to-roll irradiation process successfully incorporated in the standard 2G HTS long length production wire, low-energy proton irradiation can be a practical solution to enhance the performance of iron-based superconducting wires and tapes, as low-energy ion sources are inexpensive to operate and are readily available commercially.

Neuroblastoma is a childhood malignancy originating from the sympathetic nervous system and accounts for approximately 15% of all pediatric cancer-related deaths. To newly identify gene(s) implicated in the progression of neuroblastoma, we investigated aberrantly methylated genomic regions in mouse skin tumors. Previously, we reported that TFAP2E, a member of activator protein-2 transcription factor family, is highly methylated within its intron and its expression is strongly suppressed in mouse skin tumors compared with the normal skin. In the present study, we analyzed public data of neuroblastoma patients and found that lower expression levels of TFAP2E are significantly associated with a shorter survival. The data indicate that TFAP2E acts as a tumor suppressor of neuroblastoma. Consistent with this notion, TFAP2E-depleted neuroblastoma NB1 and NB9 cells displayed a substantial resistance to DNA damage arising from adriamycin (ADR), cisplatin (CDDP) and ionizing radiation (IR). Silencing of TFAP2E caused a reduced ADR-induced proteolytic cleavage of caspase-3 and PARP. Of note, compared with the untransfected control cells, ADR-mediated stimulation of CDK inhibitor p21WAF1 was markedly upregulated in TFAP2E‑knocked down cells. Therefore, our present findings strongly suggest that TFAP2E has a pivotal role in the regulation of DNA damage response in NB cells through the induction of p21WAF1.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligand ephrins serve crucial roles in the interactions among epithelial cells. Eph receptor/ephrin signaling regulates cell functions, including proliferation, differentiation and migration, via these cell-cell interactions. We reported previously that EPHB2, a member of the Eph receptor family, was highly expressed in chemically induced cutaneous squamous cell carcinoma (cSCC) tissues in mice. Although the higher expression level of EPHB2 has been observed in various human cancers, its roles in the development and progression of cancers are still unclear. In the present study, the functional implications of EPHB2 in the acquisition of malignant phenotypes of cSCC cells was investigated. Silencing of EPHB2 in the human cSCC cell line A431 induced epithelial-mesenchymal transition (EMT)-like morphological changes accompanied by a significant upregulation of epithelial-mesenchymal transition-associated genes such as zinc finger E-box binding homeobox 1/2. In addition, silencing of EPHB2 suppressed anchorage-independent cell growth under 3D culture conditions. Consistent with these observations, EPHB2 exhibited higher levels of expression in tumor spheres formed under 3D culture conditions than in cells cultured in adherent form, and the expression pattern of EMT markers indicated that EMT was suppressed in tumor spheres. The results of the present study indicated that EPHB2 serves a pivotal role in promoting the anchorage-independent growth of A431 cells through the suppression of EMT.

To investigate the effect of irradiation-induced defects on the superconducting characteristics of industrially produced superconductor—GdBa2Cu3O7−δ (GdBCO)—coated conductors (CCs), we irradiated the GdBCO CCs with Au ions at 2 or 10 MeV and probed them using a slow positron beam. Vacancy clusters were detected in both unirradiated and irradiated GdBCO CCs. However, the effect of ion irradiation on the GdBCO CCs was characterized as a slight reduction in the positron annihilation rate with low-momentum electrons. We also found a correlation between the annihilation rate of low-momentum electrons and the superconducting transition temperature.

// Toshinori Ozaki 1 , Mizuyo Nakamura 1 , Takehiro Ogata 1 , Meijie Sang 1, 2 , Hiroyuki Yoda 3 , Kiriko Hiraoka 3 , Meixiang Sang 1, 4 , Osamu Shimozato 1 1 Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba, Japan 2 Department of Regenerative Medicine, Graduate School of Medicine and Pharmatheutical Science, University of Toyama, Toyama, Japan 3 Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba, Japan 4 Research Center, Fourth Hospital of Hebei Medical University, Shijiazhuang, China Correspondence to: Toshinori Ozaki, email: tozaki@chiba-cc.jp Keywords: gemcitabine, mutant p53, pancreatic cancer, RUNX2, TAp63 Received: February 16, 2016 Accepted: September 25, 2016 Published: October 04, 2016 ABSTRACT Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53 -deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53 -mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2 -depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21 WAF1 and NOXA . In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53 -mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

The critical temperature Tc and the critical current density Jc determine the limits to large-scale superconductor applications. Superconductivity emerges at Tc. The practical current-carrying capability, measured by Jc, is the ability of defects in superconductors to pin the magnetic vortices, and that may reduce Tc. Simultaneous increase of Tc and Jc in superconductors is desirable but very difficult to realize. Here we demonstrate a route to raise both Tc and Jc together in iron-based superconductors. By using low-energy proton irradiation, we create cascade defects in FeSe0.5Te0.5 films. Tc is enhanced due to the nanoscale compressive strain and proximity effect, whereas Jc is doubled under zero field at 4.2 K through strong vortex pinning by the cascade defects and surrounding nanoscale strain. At 12 K and above 15 T, one order of magnitude of Jc enhancement is achieved in both parallel and perpendicular magnetic fields to the film surface., Simultaneous increase of critical temperature and critical current in superconductors is desirable for application purpose, but very difficult to realize. Here, Ozaki et al. report a simultaneous enhancement of Tc and Jc in FeSe0.5Te0.5 films with cascade defects produced by low-energy proton irradiation.

We demonstrate a twofold increase in the in-field critical current of AMSC's standard 2G coil wire by irradiation with 18-MeV Au ions. The optimum pinning enhancement is achieved with a dose of $6\times 10^{11}\ \text{Au}\ \text{ions/cm}^{2}$ . Although the 77 K, self-field critical current is reduced by about 35%, the in-field critical current (H//c) shows a significant enhancement between 4 and 50 K in fields > 1 T. The process was used for the roll-to-roll irradiation of AMSC's standard 46-mm-wide production coated conductor strips, which were further processed into standard copper laminated coil wire. The long-length wires show the same enhancement as attained with short static irradiated samples. The roll-to-roll irradiation process can be incorporated in the standard 2G wire manufacturing, with no modifications to the current process. The enhanced performance of the wire will benefit rotating machine and magnet applications.