Bladder tumour-derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms

Abstract

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore, we have examined six somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localization defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

Figure 1.

Positions of amino acid substitutions in relation to described functional domains of hamartin.

Figure 2.

(A) Immunoblot showing levels of wild-type and mutant TSC1-FLAG proteins and endogenous TSC2 in 97-1 cell lines. (B) Measurement of TSC1 RNA levels by real time RT–PCR analysis of wild-type and mutant TSC1-FLAG mRNA transcript levels in 97-1 cell lines. RT−ve is reverse transcriptase-negative control, NTC is no template control. TSC1 expression is standardized to SDHA and normalized to the 97-1 Neo cell line.

Figure 3.

(A) Immunoblot showing TSC1 and TSC2 in 97-1 Neo control and FLAG-tagged wild-type and mutant TSC1 cell line lysates immunoprecipitated with anti-TSC1 and non-specific mouse IgM antibodies. (B) Immunoblot showing expression levels of GFP-tagged wild-type and missense mutant TSC1 protein in HCV29 cell lines. Also shown are levels of S6 phosphorylation in transiently amino acid starved cells.

Figure 4.

Semi quantitative RT–PCR analysis of TSC1 RNA in normal urothelial cells and TSC1 mutant tumour samples, showing increased electrophoretic mobility of RT–PCR products amplified from cDNA from tumours containing TSC1 104C>G (A) and 314A>G (B) missense mutations. Controls are amplification reactions using RT +ve (+) and RT−ve (−) cDNA from pooled TERT-NHUC. Lower panels show HPRT RT–PCR products. Also shown are schematic representations of aberrant splicing events associated with TSC1 104C>G (A) and TSC1 314A>G (B) mutations.

Figure 5.

(A) Immunoblot showing turnover of wild-type and S35C and H68R missense TSC1 proteins in cycloheximide (CHX)-treated cells. Cells were cultured in full growth medium supplemented with 100 µg/ml CHX or DMSO vehicle alone, and lysed at time-points indicated. Tubulin is shown as a loading control. (B) Immunoblot showing stabilization of TSC1 H68R protein levels by proteasome inhibition. Cells were cultured in full growth medium and treated with 100 µg/ml CHX or DMSO vehicle alone or pre-treated with 40 µM MG-132. (C) Half-lives of wild-type TSC1 and His68Arg, Phe158Cys and His206Asp mutant proteins, as determined by ³⁵S pulse chase analysis. Cells were pulsed with 250 µCi ³⁵S in cysteine and methionine-free medium and chased in full growth medium supplemented with 200 nM cysteine and methionine. Cells were lysed at time-points as indicated and lysates immunoprecipitated with an anti-TSC1 antibody. Degradation of ³⁵S-labelled TSC1 was determined of immunoprecipitated lysates by SDS–PAGE analysis and autoradiography.

Figure 6.

Localization of wild-type TSC1 (A) and TSC1 Ser35Cys (B), Phe216Ala (C) and Thr417Ile (D) mutant proteins in amino acid-starved 97-1 cells. Cells were cultured in full growth medium on highly optically clear microscopy dishes to sub-confluence and amino acid starved for 24 h. TSC1-GFP was observed by UV microscopy of live cells. Scale bars show 100 µM.

Figure 7.

TSC1 immunostaining of HCV29 Neo (A) and TSC1 (D) cell pellets, normal ureter (negative (B) and positive (E) antibody controls) and TSC1 73–77Δ 5 (C) and TSC1 wild-type (F) bladder tumours. TSC1 staining of TSC1 missense mutant bladder tumours; TSC1 104C>G (G), 314A>G (H), 648T>A (I) and 616C>G (J–L) mutant tumours. Arrow in (H) shows normal urothelium with strong TSC1 staining adjacent to immunonegative tumour cells. (J) to (L) show TSC1 staining in tumours resected from the same patient in 2001 (J), 2003 (K) and 2004 (L). Arrows in (K) show positive TSC1 immunoreactivity in von Brunn’s nests. Scale bars show 500 µM.