Ras inhibits endoplasmic reticulum stress in human cancer cells with amplified Myc

In neuroblastoma LAN‐1 cells harboring an amplified MycN gene, disruption of cooperation between Ras and MycN proteins by the Ras inhibitor farnesylthiosalicylic acid (FTS, Salirasib) reportedly arrests cell growth. Our aim was to establish whether this is a general phenomenon. We examined the effects of FTS on gene‐expression profiles, growth and death of NCIH929 myeloma cells and K562 leukemia cells, which—like LAN‐1 cells—exhibit Myc gene amplification and harbor active Ras. Under specified conditions, FTS reduced Ras and Myc and induced cell growth arrest and death in all Myc‐amplified cell lines but not in SHEP, a neuroblastoma cell line without Myc gene amplification. Gene‐expression analysis revealed a common pattern of FTS‐induced endoplasmic reticulum (ER) stress, known as the unfolded protein response (UPR), in Myc‐amplified cells, but not in SHEP. Thus, Ras negatively regulates ER stress in cells with amplified Myc. ER stress was also inducible by dominant‐negative (DN)‐Ras or shRNA to Ras isoforms, all of which induced an increase in BIP (the master regulator of ER stress) and its downstream targets Nrf2 and eIF2α, both regulated by active p‐PERK. FTS also induced an increase in p‐PERK, while small interfering RNA to PERK reduced Nrf2 and ATF4 and rescued cells from FTS‐induced death. BIP and its downstream targets were also increased by inhibitors of MAPK p38 and MEK. Ras, acting through MAPK p38 and MEK, negatively regulates the ER stress cascades BIP/PERK/Nrf2 and eIF2α/ATF4/ATF3. These findings can explain the Ras‐dependent protection of Myc‐amplified cells from ER stress‐associated death.     

MycN sensitizes neuroblastoma cells for drug-induced apoptosis

Amplification of the MYCN gene is found in a large proportion of neuroblastoma and considered as an adverse prognostic factor. To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs we used a human neuroblastoma cell line harboring tetracycline-controlled expression of MycN. Neither conditional expression of MycN alone nor low drug concentrations triggered apoptosis. However, when acting in concert, MycN and cytotoxic drugs efficiently induced cell death. Apoptosis depended on mitochondrial permeability transition and activation of caspases, since the mitochondrion-specific inhibitor bongkrekic acid and the caspase inhibitor zVAD-fmk almost completely abrogated apoptosis. Loss of mitochondrial transmembrane potential and release of cytochrome c from mitochondria preceded activation of caspase-8 and caspase-3 and cleavage of PARP. CD95 expression was upregulated by treatment with cytotoxic drugs, while MycN cooperated with cytotoxic drugs to increase sensitivity to CD95-induced apoptosis and enhancing CD95-L expression. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression, while Bcl-2 and Bcl-X(L) protein levels remained unchanged. Since amplification of MYCN is usually associated with a poor prognosis, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.     

Rapamycin inhibits proliferation of human neuroblastoma cells without suppression of MycN

MYCN and insulin-like growth factor (IGF) system are important for the pathogenesis and development of neuroblastoma. We previously reported evidence of a direct linkage between MycN and the IGF system in KP-N-RT human neuroblastoma cells, where IGF-I induced both MycN expression at the RNA level and G1-S cell cycle progression through the IGF-I receptor (IGF-IR)/ MEK/ mitogen-activated protein kinase (MAPK) pathway (A. Misawa et al., Cancer Res, 2000; 60:64-9). Our data also showed the possibility of a potent IGF-IR downstream signal cascade that accelerates progression into the S-phase, other than the MAPK pathway. In this study, we further investigated the role of this alternative pathway in the growth of neuroblastoma cells. A phosphoinositide 3-kinase (PI3K) inhibitor wortmannin blocked IGF-I-mediated induction of MycN. Our data suggest that the inhibition of MycN by wortmannin was transmitted through the MAPK pathway. Progression of the cell cycle from G1 to S phase was inhibited up to 90% by wortmannin or rapamycin, an inhibitor of mTOR, which acts downstream of PI3K. Despite its effects on induction of MycN and on progression through S phase, wortmannin did not block proliferation of neuroblastoma cells. On the other hand, rapamycin inhibited both IGF-I-induced cell cycle progression and cell proliferation in complete medium, although it had no effect on IGF-I-mediated MycN induction. Our study indicates maintenance of cell proliferation requires mTOR function, which is independent of MycN induction in human neuroblastoma cells.     

Functional interaction of Yaf2 with the central region of MycN

MYCN is often amplified in advanced-stage neuroblastomas with the consequence of enhanced MycN protein expression. By employing the yeast two-hybrid system we found that Yaf2 binds to the central region of MycN. Binding was also seen in vitro and in vivo. Ectopically expressed Yaf2, like MycN, is localized in the nuclei of neuroblastoma cells. Endogenous Yaf2 is expressed in all three tested neuroblastoma cell lines, all of which also express MycN. Yaf2 was able to enhance MycN-mediated transactivation from an E-box promoter, deletion of the Yaf2 binding region in MycN abrogates this effect. Thus, the binding of Yaf2 to the central region of MycN is functional in mammalian cells.     

CD59 expressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma

It has been hypothesized that complement inhibitors expressed on the surface of tumor cells prevent effective immune-mediated clearance. Whereas there are in vitro data to support this hypothesis, the species-selective activity of complement inhibitors has been a hindrance to investigating the role of membrane-bound complement inhibitors in rodent models of human cancer. The CD59-positive LAN-1 human neuroblastoma cell line was significantly more sensitive to lysis by rat complement than by human complement, illustrating the species selectivity of endogenously expressed complement inhibitors. Transfection of LAN-1 cells with rat CD59, an inhibitor of the terminal cytolytic membrane attack complex, effectively protected the cells from lysis by rat complement in vitro. When LAN-1 cells stably expressing rat CD59 were inoculated into immune-deficient rats, the onset of tumor growth and the rate of tumor growth were significantly enhanced compared with those of control-transfected LAN-1 cells. These data show directly that the expression of a complement inhibitor on a tumor cell promotes tumor growth. Flow cytometric analysis revealed that the endogenous expression of decay-accelerating factor (DAF), an inhibitor of complement activation, was up-regulated on the surface of cells after in vivo growth. Of further interest, higher levels of DAF were present on CD59-transfected cells than on control-transfected cells derived from tumors. Increased DAF expression correlated with decreased complement deposition on the tumor cell surface. These results show that expression of complement inhibitors on a tumor cell has functional consequences with regard to complement deposition in vivo and indicate that CD59 can indirectly effect complement activation and C3 deposition in vivo via a link between CD59 and DAF expression.     

p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation

Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.     

Geldanamycin decreases Raf-1 and Akt levels and induces apoptosis in neuroblastomas

Neuroblastomas are the most common extracranial solid tumors of childhood. These tumors are associated with an overall poor prognosis, particularly for advanced stage disease. The benzoquinone ansamycin antibiotic, geldanamycin (GA), exhibits potent antitumor activity in certain cancer cell lines by destabilizing important signal transduction proteins (e.g., Raf-1 and Akt). The purpose of our study was to determine whether GA can alter the expression of Raf-1 and Akt, which have been shown to be critical for neuronal cell survival, and induce apoptosis of neuroblastoma cells. Human neuroblastoma cells (SH-SY5Y, SK-N-SH and LAN-1) were treated with GA for a variable period of time. Cell viability was assessed with MTT assays. Apoptosis was assessed with DNA fragmentation ELISA, TUNEL-flow cytometric assay, Western blot and caspase activities. We found that GA decreases cell viability and induces apoptosis in the SH-SY5Y human neuroblastoma cell line. These effects were mediated through activation of caspase-9 and -3, mitochondrial release of cytochrome c and subsequent PARP cleavage. GA-induced apoptosis was associated with a reduction in the level and activity of Raf-1 and Akt. The importance of these proteins was further demonstrated by induction of apoptosis in SH-SY5Y cells by a combination of U0126 (MEK1/2 inhibitor) and LY294002 (an inhibitor of PI3K). Similar to SH-SY5Y cells, other human neuroblastoma cells (SK-N-SH and LAN-1) were sensitive to the effects of GA-induced apoptosis. Taken together, our findings suggest that GA may be a novel therapeutic agent, which may be effective in the treatment of neuroblastomas.     

Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression

BACKGROUND: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. METHODS: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. RESULTS: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P =.006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. CONCLUSIONS: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.     

Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth

Objective: Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma. Methods: NGP and SH-5Y-SY cells were treated with 0–20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot. Results: Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P < 0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P < 0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression. Conclusion: Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.     

突变型[12Asp]K-ras4B基因的致瘤作用及其机制的探讨

背景与目的：有资料提示K－ras基因突变可能与子宫内膜癌的发生有关。本研究在发现子宫内膜腺癌HEC－1A细胞有K-ras基因第12密码子突变的基础上,探讨[^12Asp]K-ras4B突变基因的致瘤性及其发生机制。方法：（1）用RT－PCR方法扩增[^12Asp]K-ras4B基因全长cDNA,将此cDNA插入真核表达载体pcDI,构建pcDI-[^12Asp]K-ras4B真核表达质粒;（2）观察真核表达质粒pcDI-[^12Asp]K-ras4B转染前后细胞形态学改变,用MTT方法、^3H掺入实验、集落形成实验检测转染前后细胞体外生长的变化,以及用转基因肿瘤细胞裸鼠异种接种成瘤实验,了解[^12Asp]K-ras4B的致瘤作用。结果：（1）成功构建了真核表达质粒pcDI-[^12Asp]K-ras4B。（2）通过转染前后细胞形态学变化、体外增殖变化、软琼脂集落形成实验以及裸鼠皮下转染细胞接种实验,证明[^12Asp]K-ras4B基因具有致瘤作用。（3）将pCMV-RasN17质粒转染到pcDI-[^12Asp]K-ras4B细胞,与未转染细胞比较,发现pcDI-[^12Asp]K-ras4B细胞增生受到明显抑制。（4）分别将pCMV-RafS621A和pCMV-RafCAAX质粒转染到pcDI-[^12Asp]K-ras4B NIH3T3细胞,与未转染的细胞比较,转染pCMV-RafCAAX质粒的pcDI-[^12Asp]K-ras4B细胞增殖受到抑制,与对照组比较差异显著（P＜0．05）;转染pCMV-RafCAAX质粒的pcDI-[^12Asp]K-ras4B细胞增殖活性加强,与对照组比较差异显著（P＜0．05）。结论：（1）[^12Asp]K-ras4B基因单独作用即可诱导NIH3T3细胞瘤样转化;（2）[^12Asp]K-ras4B基因诱导的NIH3T3细胞瘤样转化是通过下游的Raf信号通路。     

Farnesyl thiosalicylic acid inhibits the growth of melanoma cells through a combination of cytostatic and pro-apoptotic effects

Novel classes of drug that interfere with the signalling of the small G-protein Ras, the so-called Ras antagonists, are showing much promise as novel anti-cancer agents. In this study, we demonstrate that the novel Ras antagonist farnesylthiosalicylic acid (FTS) inhibits the growth of Colo 853 melanoma cells through a combination of cytostatic and pro-apoptotic effects. Furthermore, these phenomena are seen under conditions of cell attachment and in the presence of serum. Treatment of Colo 853 cells with FTS led to time-dependent inhibition of constitutive Akt, retinoblastoma protein (pRB) and ERK activity, with a concurrent loss of Akt expression. Inhibition of Akt and ERK activity induces apoptosis in other human cancer cell lines. Here it is demonstrated that inhibition of Akt, or ERK and Akt in combination, leads to cell cycle arrest but not apoptosis in melanoma cells. FTS treatment was also found to upregulate activity of the stress-activated p38 MAP kinase. Inhibition of p38 MAP kinase, using the selective inhibitor SB 203580, followed by FTS treatment, significantly increased the proportion of apoptotic cells after 72 hr, possibly suggesting a modulatory role for p38 MAP kinase in FTS-induced melanoma cell apoptosis.