Lim1 oncogene as a new therapeutic target in advanced human renal cell carcinoma
Sources of Funding: INSERM, University of Strasbourg, Ligue Contre le Cancer
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.
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 Matrigel-coated 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).
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.
These results show that targeting Lim1 has therapeutic potential in this refractory disease.
INSERM, University of Strasbourg, Ligue Contre le Cancer