The loss of VHL and the subsequent dysregulation of HIF signalling are known to play a role in the development of clear cell renal cell carcinoma (ccRCC). However, a recent paper by Mikhaylova et al. (2012) has now suggested that VHL loss in ccRCC also modulates autophagy through the action of microRNA-204 (MiR-204).
MiRs are short RNA molecules which can regulate gene expression, and in 128 samples of human ccRCC, it was noted that MiR-204 levels were significantly decreased when compared to 114 matched normal kidney controls. Levels of MiR-204 were also low in the VHL-null 786-O and A498 RCC cell lines, and its expression was induced upon reintroduction of wild-type VHL. Conversely, VHL shRNA knockdown in VHL-positive Caki-1 cells led to a decrease in MiR-204 expression. Moreover, there was a significant positive correlation between MiR-204 and VHL protein levels in normal human kidneys, which strengthens the argument that VHL regulates MiR-204.
In xenograft experiments involving 786-O cells, an increase in the levels of MiR-204 led to a decrease in tumour growth and invasiveness. Further studies showed that MiR-204 was significantly cytotoxic to nutrient-deprived 786-O and A498 cells, and that this effect was abolished when an inactive mutant form of MiR-204 was used. MiR-204-dependent cytotoxicity was also lessened when nutrients were reintroduced to the culture medium, and this finding prompted the authors to investigate starvation-induced autophagy.
Consequently, it was seen by transmission electron microscopy that an increase in MiR-204 in 786-O cells led to a decrease in the number of autophagic vesicles observed. These vesicles were also smaller and less developed when compared to untreated controls. LC3-II is a marker of active autophagy, and when using western blot and immunofluorescence, the levels of LC3B-II were also significantly lower in 786-O and A498 cells treated with MiR-204. In silico analysis identified a MiR-204 binding site in the 3’UTR of LC3B, and when this site was mutated in a luciferase reporter assay, it was immune to repression by MiR-204 (in contrast to the wild-type 3’UTR of LC3B). Furthermore, a significant negative correlation was observed between MiR-204 and LC3B protein levels in human ccRCC tumours, further indicating that LC3B-dependent autophagy may be a direct target of MiR-204.
Using 786-O and A498 cells in xenograft experiments, shRNA knockdown of LC3B significantly inhibited tumour growth when compared to shRNA controls. There was also a significant positive correlation between LC3B levels and tumour grade in human ccRCC tumours, with MiR-204 showing a significant negative relationship. These results suggest that MiR-204-mediated inhibition of autophagy may suppress the development of ccRCC.
Autophagy is a process in which the cell can recycle its components under conditions of nutrient deprivation. However, it can also be subverted during tumourigenesis, as has been suggested in another study by Parkhitko et al. (2011) (as detailed in a previous blog post). Could a similar process be taking place in BHD syndrome, and could it be exploited to develop novel treatments?
- Mikhaylova O, Stratton Y, Hall D, Kellner E, Ehmer B, Drew AF, Gallo CA, Plas DR, Biesiada J, Meller J, & Czyzyk-Krzeska MF (2012). VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma. Cancer cell, 21 (4), 532-46 PMID: 22516261
- Parkhitko A, Myachina F, Morrison TA, Hindi KM, Auricchio N, Karbowniczek M, Wu JJ, Finkel T, Kwiatkowski DJ, Yu JJ, & Henske EP (2011). Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent. Proceedings of the National Academy of Sciences of the United States of America, 108 (30), 12455-60 PMID: 21746920