MiR-21 enhances melanoma invasiveness via inhibition of tissue inhibitor of metalloproteinases 3 expression: in vivo effects of MiR-21 inhibitor.

Metastatic melanoma is the most aggressive form of this cancer. It is important to understand factors that increase or decrease metastatic activity in order to more effectively research and implement treatments for melanoma. Increased cell invasion through the extracellular matrix is required for metastasis and is enhanced by matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinases 3 (TIMP3) inhibits MMP activity. It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma. It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby enhance the invasiveness of melanoma cells. miR-21 over-expression in the melanoma cell lines WM1552c, WM793b, A375 and MEL 39 was accomplished via transfection with pre-miR-21. Immunoblot analysis of miR-21-overexpressing cell lines revealed reduced expression of TIMP3 as compared to controls. This in turn led to a significant increase in the invasiveness of the radial growth phase cell line WM1552c and the vertical growth phase cell line WM793b (p < 0.05), but not in the metastatic cell lines A375 or MEL 39. The proliferation and migration of miR-21 over-expressing cell lines was not affected. Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cell lines, mimicking the effects of miR-21 over-expression. Treatment of tumor cells with a linked nucleic acid antagomir to miR-21 inhibited tumor growth and increased tumor expression of TIMP3 in vivo in 01B74 Athymic NCr-nu/nu mice. Intra-tumoral injections of anti-miR-21 produced similar effects. This data shows that increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.

Interleukin-29 binds to melanoma cells inducing Jak-STAT signal transduction and apoptosis.

Interleukin-29 (IL-29) is a member of the type III IFN family that has been shown to have antiviral activity and to inhibit cell growth. Melanoma cell lines were tested for expression of the IL-29 receptor (IL-29R) and their response to IL-29. Expression of IL-28R1 and IL-10R2, components of IL-29R, was evaluated using reverse transcription-PCR. A combination of immunoblot analysis and flow cytometry was used to evaluate IL-29-induced signal transduction. U133 Plus 2.0 Arrays and real-time PCR were used to evaluate gene expression. Apoptosis was measured using Annexin V/propridium iodide staining. In situ PCR for IL-29R was done on paraffin-embedded melanoma tumors. Both IL-28R1 and IL-10R2 were expressed on the A375, 1106 MEL, Hs294T, 18105 MEL, MEL 39, SK MEL 5, and F01 cell lines. Incubation of melanoma cell lines with IL-29 (10-1,000 ng/mL) led to phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2. Microarray analysis and quantitative reverse transcription-PCR showed a marked increase in transcripts of IFN-regulated genes after treatment with IL-29. In the F01 cell line, bortezomib-induced and temozolomide-induced apoptosis was synergistically enhanced following the addition of IL-29. In situ PCR revealed that IL-10R2 and IL-28R1 were present in six of eight primary human melanoma tumors but not in benign nevi specimens. In conclusion, IL-29 receptors are expressed on the surface of human melanoma cell lines and patient samples, and treatment of these cell lines with IL-29 leads to signaling via the Jak-STAT pathway, the transcription of a unique set of genes, and apoptosis.