Melanoma is a highly metastatic cancer, but the mechanisms that underlie this phenotype are not completely understood. Colin Goding and colleagues now show that the microphthalmia-associated transcription factor, MITF, functions in a dynamic fashion to promote either the proliferation or metastasis of melanoma cells.
MITF is a potent oncogene that is required for the proliferation of melanoma cells with activated BRAF. However, MITF is also implicated in promoting melanocyte differentiation, and can induce G1 cell-cycle arrest. To investigate the functions of MITF in vitro, Goding and colleagues depleted MITF in 501mel melanoma cells using short interfering RNA (siRNA). MITF depletion led to G1 cell-cycle arrest, and MITF-depleted cells seemed more rounded. Immunostaining revealed a change in the F-actin organization in these cells, explaining their change in cell shape. Western blotting showed that the depletion of MITF leads to the increased expression of the cyclin-dependent kinase inhibitor, p27 (encoded by CDKN1B). Double siRNA knockdown of MITF and CDKN1B rescued the G1 block. However, ectopic expression of p27 in MITF-expressing 501mel cells did not result in altered F-actin organization. So, MITF induces G1 arrest through a p27-dependent pathway and controls F-actin organization through a p27-independent pathway.
Although the level of p27 was substantially increased in MITF-depleted cells, there was no corresponding significant increase in CDKN1B mRNA levels, indicating that the loss of MITF stabilizes p27. The stability of p27 is controlled by the F-box protein SKP2, which is regulated indirectly by the Diaphanous-related formin, DIAPH1, a Rho effector protein that regulates F-actin polymerization. The depletion of MITF led to a decrease in the level of DIAPH1 mRNA and protein. Furthermore, ectopic expression of DIAPH1 restored both normal F-actin organization and p27 expression in MITF-depleted cells. MITF binding sites were identified in the promoter of DIAPH1, indicating that MITF directly controls DIAPH1 expression, thereby regulating F-actin organization and cell-cycle progression.
The authors went on to investigate the effect of MITF expression in an invasive melanoma cell line, SkMel28. Ectopic expression of MITF inhibited the ability of these cells to form tumours when they were injected into nude mice, and decreased their invasiveness in matrigel assays. Moreover, MITF depletion in these cells led to increased metalloproteinase expression, a frequent characteristic of invasiveness. Importantly, these MITF-depleted invasive cells have high levels of p27 and are therefore not dividing, making them more likely to be resistant to chemotherapy.
The authors conclude that MITF, by acting through DIAPH1, is a key determinant of invasive potential and both cell-cycle entry and exit in melanoma cells. They suggest that MITF might respond to various stimuli from the tumour microenvironment to dictate the differentiation, proliferation and metastatic ability of melanoma cells. This would represent a new mechanism of cancer progression that does not arise from a stably-inherited genetic mutation.
ORIGINAL RESEARCH PAPER
- Carreira, S. et al. MITF regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev. 15 December 2006 (doi:doi: 10.1101/gad.405406) | Article |