Last week’s blog post described a clear cell renal cell carcinoma (ccRCC) paper which suggests that microRNA-204 (MiR-204) acts as a VHL-regulated tumour suppressor through the inhibition of LC3B-associated autophagy (Mikhaylova et al., 2012).
In this study, immunofluorescence experiments showed that an increase in MiR-204 led to an accumulation of factors involved in the initiation of autophagy in nutrient-deprived VHL-null 786-O cells (but not in VHL-positive cells), suggesting that MiR-204 may inhibit autophagosome maturation.
In addition, the levels of MiR-204 in 786-O cells were not affected by hypoxia or siRNA-mediated knockdown of HIF2α, indicating that this microRNA is regulated independently of hypoxia-inducible factor (HIF) signalling (which is often dysregulated in ccRCC as a direct result of VHL-loss).
Using quantitative RT-PCR and western blot, the authors also found that reintroduction of VHL in VHL-null 786-O and A498 cells increased the mRNA and protein levels of a lesser-known paralogue of LC3B, known as LC3C. Conversely, shRNA knockdown of VHL in VHL-positive Caki-1 cells reduced LC3C protein expression. Quantitative RT-PCR also demonstrated that LC3C is sensitive to HIF, as shRNA knockdown of HIF2α induced LC3C mRNA expression, and activation of HIF2α (with a prolyl hydroxylase inhibitor) reduced the levels of LC3C mRNA.
Considering that LC3B-mediated autophagy appears to promote ccRCC progression (as detailed in last week’s blog post), what are the effects of LC3C? Mikhaylova et al. noted that shRNA knockdown of LC3C in VHL-positive 786-O and A498 cells led to an increase in the formation of small tumours in mouse xenograft experiments. Furthermore, western blot analysis of 8 human ccRCC samples showed an overall decrease in LC3C protein expression when compared to matched normal kidney specimens. Taken together, these results indicate that LC3C may be involved in tumour suppression.
What this work illustrates is that factors involved in autophagy appear to both inhibit and promote tumourigenesis, as discussed by Professor Eileen White at the American Association for Cancer Research Annual Meeting 2012 (which is described in a recent conference report). Moreover, VHL seems to regulate access to nutrients from both intra- and extra-cellular sources through autophagy and angiogenesis, by both HIF-dependent and HIF-independent pathways. Consequently, specific autophagy inhibitors could be used in combination with alternative therapies, a strategy which may also be applicable to BHD syndrome.
- 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