WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (∼30-300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent.

Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells.

The rat hepatoma-human fibroblast hybrid cell line WIF-B9 stably exhibits the structural and functional characteristics of normal differentiated hepatocytes. The abilities of these cells to synthesize bile acids and amidate them with glycine and taurine were investigated. The release of bile acids into the culture media over 72 h was assessed by gas chromatography-mass spectrometry. WIF-B9 cells were able to synthesize bile acids (1.10+/-0.17 nmol/mg protein) but less efficiently than rat hepatocytes in primary culture (2.19+/-0.19 nmol/mg protein; P<0.01). The patterns of major bile acid species produced by both types of cells were also different. Cholic acid (CA; 72%) and beta-muricholic acid (19%) were the major bile acids produced by rat hepatocytes, while chenodeoxycholic acid (CDCA) accounted for only 4.5% of total bile acids. In contrast, muricholic acids were absent, while CA (62%) and CDCA (34%) were the most abundant bile acids synthesized by WIF-B9 cells. Using reverse transcription-polymerase chain reaction and gene- and species-specific primers for key enzymes involved in bile acid synthesis, the expression of human, but not rat, orthologues of CYP7A1, CYP27, CYP8B and CYP7B1 was found in WIF-B9 cells. Induction of cell stress by serum deprivation did not change the amount of total bile acids synthesized by these cells, but an inversion of the CA-to-CDCA ratio from 1.8 to 0.3 together with a marked increase in the proportion of intermediate metabolites related to the acidic pathway was found. Using 500 microM radiolabeled CA and 2 mM of taurine or glycine, the ability to amidate CA over 48 h was determined by high performance liquid chromatography. Rat hepatocytes conjugated more than 90% CA with either amino acid, whereas this ability was very poor (< 2%) in WIF-B9 cells. Regarding the expression of enzymes and the products of bile acid synthesis, it may be concluded that the human phenotype predominates over that of the rat in WIF-B9 cells. Moreover, these cells are almost completely unable to further conjugate primary bile acids, which facilitates the manipulation of these steroids in analytical procedures. These characteristics make WIF-B9 cells a suitable in vitro model to carry out studies on bile acid synthesis by 'human-like' metabolic pathways.

Molecular cloning and characterization of the 5' region of the mouse trkA proto-oncogene.

The trkA proto-oncogene encodes a high-affinity NGF receptor that is essential for the survival, differentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurodegeneration, tumor progression and oncogenesis. We have cloned and characterized the 5' region of the mouse trkA gene and have identified its promoter. trkA promoter sequences are GC-rich, lack genuine TATA or CAAT boxes, and are contained within a CpG island which extends over the entire first coding exon. The mouse trkA transcription start site is located 70/71 bp upstream to the AUG translation initiation codon. Sequence analysis showed that the gene encoding the insulin receptor-related receptor, IRR, is located just 1.6 kbp upstream to the trkA gene and is transcribed in the opposite direction. We have used trkA-CAT transcriptional fusions to study trkA promoter function in transient transfection experiments. RNase protection assays and CAT protein ELISA analyses showed that a 150 bp long DNA segment, immediately upstream to the start site, is sufficient to direct accurate transcription in trkA-expressing cells. Dissection of this fragment allowed us to identify a 13 bp cis-regulatory element essential for both promoter activity and cell-type specific expression. Deletion of this 13 bp segment as well as modification of its sequence by site-directed mutagenesis led to a dramatic decline in promoter activity. Gel mobility shift assays carried out with double-stranded oligonucleotides containing the 13 bp element revealed several specific DNA-protein complexes when nuclear extracts from trkA-expressing cells were used. Supershift experiments showed that the Sp1 transcription factor was a component of one of these complexes. Our results identify a minimal trkA gene promoter, located very close to the transcription start site, and define a 13 bp enhancer within this promoter sequence.

Proximal promoter sequences mediate cell-specific and elevated expression of the favorable prognosis marker TrkA in human neuroblastoma cells.

The nerve growth factor receptor, TrkA, has a critical role in the survival, differentiation, and function of neurons in the peripheral and central nervous systems. Recent studies have demonstrated a strong correlation between abundant expression of TrkA and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and differentiation of neuroblastoma tumor cells and may be an important therapeutic target in the treatment of this disease. In the present study, we have examined the mechanistic basis for TrkA gene expression in human neuroblastoma cells. Northern blotting and nuclear run-on analyses demonstrated that transcription is a primary determinant of both cell-specific and variable expression of the TrkA gene in neuroblastoma cell lines that express it to different degrees. Cell-specific and variable transcription in neuroblastoma cells was recapitulated by transient transfection of TrkA promoter-luciferase reporter constructs, and regulatory sequences mediating these processes were localized to a 138-base pair region lying just upstream of the transcription initiation region. This neuroblastoma regulatory region formed multiple DNA-protein complexes in gel shift assays that were highly enriched in neuroblastoma cells exhibiting abundant TrkA expression. Thus, TrkA-positive neuroblastoma cells are distinguished by differential expression of putative transcription factors that ultimately may serve as targets for up-regulating TrkA expression in tumors with poor prognosis.