VHL genetic alterations do not affect the production of HIF-α in clear-cell renal cell carcinoma (ccRCC). However, their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expression are currently unknown. In a recent study, Damjanovic et al. (2016) evaluated the impact of VHL genetic alterations and HIF-α production on the expression of TSC proteins and Hsp90 in 47 sporadic ccRCCs and corresponding normal tissues.
The authors found somatic mutations in VHL gene in 31/47 of the ccRCC samples. Most of these mutations were frameshifts caused by intragenic deletions and duplications. Monoallelic inactivation (intragenic mutation or loss of heterozygosity (LOH)) was found in 10/47 and biallelic inactivation (intragenic mutation plus LOH) in 27/47 of ccRCCs. Altogether, 37/47 of ccRCC samples had alteration in VHL region.
mRNA expression of HIF-1α and HIF-2α was similar in tumours and normal tissues and VHL independent. In addition, the abundance of HIF-α mRNAs was similar in HIF-1α positive and negative tumours.
Western blot analysis showed that HIF-1α and HIF-2α were overexpressed in 10/47 and 21/47 of tumours compared to normal tissue. In 30/47 of tumours HIF-1α expression was reduced in comparison to control and in 16/47 of ccRCCs samples HIF-1α was undetectable. Multiple tests revealed that only tumours with loss of HIF-1α overexpressed HIF-2α. Tumorous production of HIF-α proteins was unaffected by alterations in VHL. These results suggest that the expression of these proteins is post-transcriptionally regulated.
TSC1 expression on tumours was affected by both VHL functional status and HIF-1α production. TSC1 expression was reduced in tumours with monoallelic VHL inactivation compared to control and tumours with biallelic inactivation. This lower expression was found more frequently among tumours with loss of HIF-1α. In addition, Fuhrman’s nuclear grade was higher in tumours overexpressing TSC1.
VHL functional status and HIF-1α production did not affect Hsp90 expression in ccRCCs, suggesting that its role in HIF-α stabilization is maintained.
In addition to the western blot analysis, immunostaining revealed that expression of both TSC1 and TSC2 was reduced in tumours, but only TSC1 expression was dependent on VHL functional status. TSC2 expression was reduced in all tumours while TSC1 only in tumours with VHL alterations. In addition, ccRCCs with loss of HIF-1α showed reduced immunostaining for TSC1. These results suggest that HIF-1α could be involved in positive regulation of TSC1 expression in these tumours and that wild-type pVHL negatively regulates TSC1 expression.
Tumour size positively correlated with tumour grade and TSC1 expression. In VHL altered ccRCCs tumour diameter correlated with nuclear grade, TSC1 and Hsp90 expression and with the HIF-2α expression ratio between control and tumour. In stepwise multivariate linear regression analysis, a significant portion of variability in tumour diameter could be explained by variations in nuclear grade, TSC1 expression and HIF-2α expression ratio. In ccRCCs with VHL alterations, a significant portion of variability in the tumour size can be explained by Fuhrman’s grade, TSC1 expression and HIF-2α overexpression. Because nucleolar size depends on mTORC1 activity (Fuhrman et al., (1982), Li et al., (2009)), the authors results suggest a TSC1 dependency on pVHL.
In summary, while TSC2 is broadly downregulated in ccRCC, TSC1 expression is reduced in two subsets of these tumours, those with monoallelic VHL gene inactivation and those with both low HIF-1α and high HIF-2α expression. The effect of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumours, suggests that pVHL regulates TSC1. These results are important for future studies on ccRCC where reduced expression and imbalances in TSC1/TSC2 expression should be considered in developing future therapies.
- Damjanovic SS, Ilic BB, Beleslin Cokic BB, Antic JA, Bankovic JZ, Milicevic IT, Rodic GS, Ilic DS, Todorovic VN, Puskas N, & Tulic CD (2016). Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma. Experimental and molecular pathology, 101 (3), 323-331 PMID: 27845047