Valérian Dormoy

Role of the RNA-binding protein HuR in human renal cell carcinoma

Human conventional renal cell carcinoma (CRCC) remains resistant to therapy. The RNA-binding protein HuR regulates the stability and/or translation of multiple messenger RNAs involved in malignant transformation. In this study, we aimed to evaluate the potential role of HuR in this pathology. Using seven human CRCC cell lines expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as well as 15 normal/renal cell carcinoma tumor pairs, we showed that HuR is overexpressed in all tumors independently of the VHL status. Futhermore, HuR cytoplasmic presence appears to be more common in early tumor stages, suggesting a role in tumor promotion. We then assessed the effect of HuR knockdown using small interfering RNA in cultured cell and in tumor-bearing mice. Both in vitro and in vivo, we observed that cell growth was inhibited by 60% and that this effect was obtained through an inhibition of cell proliferation and an induction of cell apoptosis. Finally, we found that expression of vascular endothelium growth factor, tumor growth factor-beta and of the hypoxia-induced transcription factor-2alpha as well as the constitutive activation of the oncogenic phosphoinositide 3-kinase/Akt, nuclear factor-kappaB and mitogen-activated protein kinase pathways were decreased in HuR-depleted cells and tumors. All these results suggest a pivotal role for HuR in human CRCC.

Par6γ is at the mother centriole and controls centrosomal protein composition through a Par6α-dependent pathway

Summary The centrosome contains two centrioles that differ in age, protein composition and function. This non-membrane bound organelle is known to regulate microtubule organization in dividing cells and ciliogenesis in quiescent cells. These specific roles depend on protein appendages at the older, or mother, centriole. In this study, we identified the polarity protein partitioning defective 6 homolog gamma (Par6&ggr;) as a novel component of the mother centriole. This specific localization required the Par6&ggr; C-terminus, but was independent of intact microtubules, the dynein/dynactin complex and the components of the PAR polarity complex. Par6&ggr; depletion resulted in altered centrosomal protein composition, with the loss of a large number of proteins, including Par6&agr; and p150Glued, from the centrosome. As a consequence, there were defects in ciliogenesis, microtubule organization and centrosome reorientation during migration. Par6&ggr; interacted with Par3 and aPKC, but these proteins were not required for the regulation of centrosomal protein composition. Par6&ggr; also associated with Par6&agr;, which controls protein recruitment to the centrosome through p150Glued. Our study is the first to identify Par6&ggr; as a component of the mother centriole and to report a role of a mother centriole protein in the regulation of centrosomal protein composition.

MP60-09 LIM1 ONCOGENE AS A NEW THERAPEUTIC TARGET IN ADVANCED HUMAN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Clear cell renal cell carcinoma (CCC) is resistant to therapies. We and others have shown the oncogenicity of various signaling pathways/markers including the PI3K/Akt, NF-kB, MAPK, sonic hedgehog (SHH)-Gli and Notch pathways and Pax2 transcription factor. These are also pathways/markers involved in nephrogenesis leading us to hypothesize that tumor cells hijack developmental signaling pathways/markers for their own growth. Among Gli targets, we have identified the developmental Lim1 transcription factor as a new oncogene in CCC, regulating tumor growth. Preliminary results also suggested a role in metastasis development. Here, we subsequently investigated whether Lim1 has a role in advanced CCC. METHODS: Human 786-0, A498 (VHL-) and Caki2, ACHN (VHL+) cells were used. No chemical inhibitor of Lim1 is available. We thus investigated its role in tumor invasion using siRNA and Lim1 expressing vector. In vitro, Lim1 effect on cell motility, migration and invasion was studied in cells transiently transfected with Lim1 siRNAs for 24-96h, by wound healing assay, and using uncoated and Matrigelcoated Boyden chamber respectively. We assessed the expression of various proteins involve in cell movements after Lim1 silencing by Western blot and PCR. We also analyzed Lim1 expression in 8 metastatic samples (lymph node and adrenal metastases). To study the impact of Lim1 in vivo, we developed a model that we calibrated for metastasis spread qualitatively and quantitatively through injection of 50 000 tumor cells into the tail vein of nude mice. Untransfected cells and cells transfected with a vector expressing Lim1 or Lim1 siRNA were used. 10 days after cell injection mice were euthanized and organs were harvested for metastases analysis and molecular studies using the HTG EdgeSeq Oncology Biomarker Panel Assay (2560 genes). RESULTS: Lim1 expression was downregulated by > 95% after siRNAs transfection. In all cell lines, the depletion of Lim1 inhibited not only cell movements by up to 50 % in a time-dependent manner, but also the expression of various proteins including Fibronectin, MMP8/9, Paxillin, and CXCR4. Lim1 was found in all metastatic samples and the corresponding primary tumor. As expected, in vivo, lung and liver metastasis developed within 10 days post-injection. We are currently analyzing organs in terms of size and number of metastases. First results suggest that Lim1 is involved in metastasis development. Upcoming experiments will define the molecular mechanisms of such effects. CONCLUSIONS: These results show that targeting Lim1 has therapeutic potential in this refractory disease.