Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-B (NF-B) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-B, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-B, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

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Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein.The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.