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[Cancer Research 66, 7934-7938, August 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Tsc1 Haploinsufficiency without Mammalian Target of Rapamycin Activation Is Sufficient for Renal Cyst Formation in Tsc1+/– Mice

Catherine Wilson, Cleo Bonnet, Carol Guy, Shelley Idziaszczyk, James Colley, Vikki Humphreys, Julie Maynard, Julian R. Sampson and Jeremy P. Cheadle

Department of Medical Genetics, Cardiff University, Cardiff, United Kingdom

Requests for reprints: Jeremy P. Cheadle, Department of Medical Genetics, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom. Phone: 44-29-20742652; Fax: 44-29-20746551; E-mail: cheadlejp{at}cardiff.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tuberous sclerosis complex (TSC) is caused by mutations in either the TSC1 or TSC2 gene. Both genes are generally considered to act as tumor suppressors that fulfill Knudson's "two-hit hypothesis" and that function within the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin (mTOR) pathway. We previously generated Tsc1+/– mice that are predisposed to renal cysts, which develop into cystadenomas and renal cell carcinomas. Here, we identified somatic Tsc1 mutations (second hits) in ~80% of cystadenomas and renal cell carcinomas, but only 31.6% of cysts from Tsc1+/– mice (P < 0.0003), raising the possibility that haploinsufficiency for Tsc1 plays a role in cyst formation. Consistent with this proposal, many cysts showed little or no staining for phosphorylated mTOR (53%) and phosphorylated S6 ribosomal protein (37%), whereas >90% of cystadenomas and renal cell carcinomas showed strong staining for both markers (P < 0.0005). We also sought somatic mutations in renal lesions from Tsc1+/– Blm–/– mice that have a high frequency of somatic loss of heterozygosity, thereby facilitating the detection of second hits. We also found significantly less somatic mutations in cysts as compared with cystadenomas and renal cell carcinomas from these mice (P = 0.017). Our data indicate that although activation of the mTOR pathway is an important step in Tsc-associated renal tumorigenesis, it may not be the key initiating event in this process. (Cancer Res 2006; 66(16): 7934-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of benign hamartomatous growths in multiple organs and tissues, including the brain, heart, skin, lungs, and kidneys (1). About 60% of cases with TSC are sporadic, representing new mutations, and TSC occurs in at least 1 in 10,000 live births without apparent ethnic clustering (2). Patients with TSC harbor a wide variety of germ-line mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13.3 (reviewed in ref. 3). Both genes are considered to act as tumor suppressors and several studies have identified somatic mutations in the wild-type TSC1 or TSC2 alleles (so-called "second hits") in TSC-associated renal angiomyolipomas (4), in accordance with Knudson's "two-hit" hypothesis of tumorigenesis (5). However, somatic mutations seem to be very uncommon in the majority of TSC-associated brain lesions (4), although physiologically inappropriate phosphorylation of the TSC2 gene product, tuberin, may act as a second hit in some of these (6).

Hamartin, the TSC1-encoded protein, and tuberin form a complex in vivo (7). Studies in Drosophila and mammals have shown that this complex functions within the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway and regulates nutrient and growth factor signaling to mTOR (reviewed in refs. 8, 9). Activation of growth-promoting receptor tyrosine kinases stimulates PI3K and the serine/threonine kinase Akt. Akt subsequently phosphorylates tuberin, down-regulating its GTPase-activating protein activity and allowing the buildup of GTP-bound Rheb, which then up-regulates mTOR. Activation of mTOR leads to phosphorylation of S6 ribosomal protein (S6) and eIF-4E binding protein 1 thereby promoting their roles in cell growth.

Several groups have generated mouse lines with constitutively inactivated Tsc1 (Tsc1+/–; refs. 10–12) and Tsc2 (Tsc2+/–; refs. 13, 14) alleles and a naturally occurring rat model of Tsc2 inactivation (the "Eker rat") has also been identified (15). All of these models are predisposed to renal tumors and Eker rats also develop pituitary adenomas, uterine leiomyomas and leiomyosarcomas, splenic hemangiomas, and at low frequency, a variety of brain lesions including lesions resembling human TSC-associated subependymal nodules and cortical tubers (16, 17). Renal lesions in Tsc1+/– and Tsc2+/– mice and Eker rats include simple cysts, cystadenomas (cysts with branching papillary projections into the lumen), and renal cell carcinomas. The distribution in the size of the lesions, with simple cysts being smaller and more numerous than cystadenomas and renal cell carcinomas, the preponderance of cysts in younger animals, and the morphologic continuity of these lesions support a model of progression from cyst through cystadenoma and into renal cell carcinoma (12). Somatic loss of heterozygosity (LOH) at the Tsc2 locus was found in only 21% (4 of 19) of single altered renal tubules from Eker rats (18), raising the possiblility that although two hits are present in some preneoplastic lesions, haploinsufficiency for Tsc2 may be sufficient for their formation. Here, we investigated the role of Tsc1 haploinsufficiency in Tsc-associated renal tumorigenesis by examining cysts, cystadenomas, and renal cell carcinomas from Tsc1+/– and Tsc1+/– Blm–/– (Bloom-deficient) mice using a combination of complementary mutation-screening strategies and immunohistochemistry.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal care, genotyping, necropsy, and pathology. All procedures with animals were carried out in accordance with Home Office guidelines. Kidneys were collected from Tsc1+/– and Tsc1+/– Blm–/– mice as previously described (12, 19). Half of each kidney was snap frozen in liquid nitrogen–cooled isopentane for laser capture microdissection and the other half was processed into paraffin wax and sectioned at 4 µm for H&E or immunohistochemistry. Immunohistochemistry with anti-mTOR, anti–phospho-mTOR (Ser2448), anti–S6 ribosomal protein, and anti–phospho-S6 ribosomal protein (Ser240/244; Cell Signalling Technologies, Danvers, MA) was done as previously described (12).

Somatic mutation analysis. DNA was extracted from renal lesions after laser capture microdissection from kidneys of 55 Tsc1+/– and 31 Tsc1+/– Blm–/– mice. LOH at the Tsc1 locus was assayed by simultaneous amplification of both the wild-type (Exon 8F 5'-FAM-TGCCTGGAAGCCCAGGAAGGT-3' and Exon 8R 5'-CTGCAGGGCCCATGGTGGTT-3') and mutant (IRES F 5'-FAM-TAACGTTACTGGCCGAAG-3' and IRES R 5'-GTCGCTACAGACGTTGTT-3') alleles in a 25-cycle PCR reaction with 3 ng of DNA (multiple sections of the same lesion were sometimes required to provide sufficient yields of DNA). Two microliters of PCR products were mixed with an ABI GS500 internal size standard and formamide loading buffer and run on an ABI3100 Genetic Analyzer. Results were analyzed with Genescan v.3.7 software. DNA extracted from eight different normal tissue sections from Tsc1+/– mice was used to normalize the assay (comparison of wild-type/mutant allele peak heights). LOH was defined as a change in wild-type/mutant peak ratios of <0.67 or ≥1.3. To search for intragenic somatic Tsc1 mutations, the entire Tsc1 open reading frame (ORF) was amplified as 23 fragments (primer sequences are available on request) and sequenced directly. Comparison between numbers of somatic mutations was done with Fisher's exact test.

Phosphorylated-mTOR and phosphorylated-S6 staining. Renal lesions were identified after H&E staining and adjacent sections were stained with antibodies to phosphorylated mTOR (p-mTOR) or phosphorylated S6 (p-S6; kidneys from 36 Tsc1+/– and 30 Tsc1+/– Blm–/– mice were used for the p-mTOR analyses and 44 Tsc1+/– mice were used for the p-S6 analyses). Staining intensities were ranked by two independent investigators as little/absent or strong (there was 70.4% and 81.2% concordance in staining estimates for p-mTOR and p-S6, respectively). Comparisons between staining grades were done with the Mann-Whitney confidence interval test.

Determination of cyst and cell size. Total cyst area was measured with Motic image plus 2.0 software in 17 p-S6 positively stained cysts and 9 p-S6 negatively stained cysts, and compared by ANOVA. Cell areas were estimated by measuring the height and width of 20 adjacent cells within 12 p-S6 positively stained cysts, 9 p-S6 negatively stained cysts, 6 cystadenomas and 6 renal cell carcinomas, and 20 normal tubule cells selected at random within the same kidneys. Sizes were compared by ANOVA followed by a Tukey test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Somatic Tsc1 mutations are not abundant in renal cysts from Tsc1+/– mice. DNA was extracted from 19 renal cysts, 49 renal cystadenomas, and 65 renal cell carcinomas from Tsc1+/– mice after laser capture microdissection. We sought somatic Tsc1 mutations in DNA from these lesions by LOH analyses and by direct sequencing of the entire Tsc1 ORF. In total, we identified 97 of 133 (72.9%) of anticipated somatic Tsc1 mutations, which included 70 LOH events and 27 intragenic mutations. Interestingly, in 68.4% (13 of 19) of cysts, we did not identify somatic Tsc1 mutations, as compared with only 20.4% (10 of 49) of cystadenomas and 20.0% (13 of 65) of renal cell carcinomas (Table 1 ; Fig. 1 ). Although it is likely that we missed some somatic mutations lying outside of the ORF and would not have detected inactivation of alleles by epigenetic silencing, we observed significantly fewer second hits in cysts as compared with cystadenomas (P = 0.0003) and renal cell carcinomas (P = 0.0001).


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  		Table 1. Somatic Tsc1 mutations in renal lesions from Tsc1+/– mice

 

Figure 1
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  		Figure 1. Examples of somatic mutations identified in cysts from Tsc1+/– mice. A, an example of LOH in a renal cyst. Arrow, loss of the wild-type Tsc1 allele in the cyst (bottom) but not in adjacent normal tissue (top). WT, wild-type allele; Mut, mutant allele. B, an example of an intragenic nonsense mutation (S163X) in a renal cyst. The C-to-A substitution at nucleic acid position 488 was identified in the cyst (bottom; arrow) but not in the adjacent normal tissue (top).

 
The mTOR pathway is activated in only a proportion of renal cysts. We investigated whether the low level of somatic Tsc1 mutations in cysts correlated with low levels of mTOR activation. We assessed the expression levels of phosphorylated (activated) (p-)mTOR in 45 renal cysts, 51 renal cystadenomas, and 27 renal cell carcinomas from Tsc1+/– mice by immunohistochemistry. In total, 76.4% (94 of 123) of lesions showed strong staining for p-mTOR. However, 53.3% (24 of 45) of cysts showed little or no p-mTOR staining, compared with only 3.9% (2 of 51) of cystadenomas and 11.1% (3 of 27) of renal cell carcinomas, the majority (>88.9%) of which showed strong staining (Table 2 ; Fig. 2 ). We also assessed the expression levels of p-S6 in 54 renal cysts, 60 renal cystadenomas, and 38 renal cell carcinomas. One hundred twenty-five of 152 (82.2%) lesions showed strong staining for p-S6. However, 37.0% (20 of 54) of cysts showed little or no p-S6 staining, compared with only 5.0% (3 of 60) of cystadenomas and 10.5% (4 of 38) of renal cell carcinomas, the majority (>89.5%) of which showed strong staining (Table 2; Fig. 2). Therefore, significantly fewer cysts seem to have p-mTOR and p-S6 activation compared with cystadenomas and renal cell carcinomas (P < 0.00001).


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  		Table 2. p-mTOR and p-S6 immunohistochemistry analyses of renal lesions from Tsc1+/– mice

 

Figure 2
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  		Figure 2. Immunohistochemistry of renal lesions from Tsc1+/– mice with anti-p-mTOR (A) and anti-p-S6 (B) antibodies. A, left, a renal cell carcinoma (top right-hand corner) shows intense brown staining with p-mTOR (boxed region enlarged in middle image), whereas a cyst (bottom left-hand corner) shows no staining (boxed region enlarged in right image). B, left, some cysts (right-hand side) showed staining with p-S6 (boxed region enlarged in middle image), whereas others (left-hand side) showed no staining (boxed region enlarged in right image). Control staining with S6 and mTOR antibodies showed consistent staining throughout the kidney and all lesions studied (data not shown). Cysts that did not stain for either p-mTOR or p-S6 were rarely adjacent to renal cell carcinomas (which might have contributed to their formation). Bar, 200 µm.

 
Detailed analysis of renal cysts. We determined the pattern of p-S6 activation throughout individual renal cysts from Tsc1+/– mice by staining consecutive serial sections. Seventeen of 26 (65%) cysts studied in this way showed consistently strong p-S6 staining and the remaining 9 (35%) showed consistently little or no staining in every serial section (Fig. 3 ). Three of the cysts with little or no p-S6 staining had some sections in which single cells displayed strong positivity (e.g., in Fig. 3D); however, this pattern was also seen in some normal tubular epithelial cells.


Figure 3
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  		Figure 3. Immunohistochemistry of consecutive serial sections of renal cysts from Tsc1+/– mice with a p-S6 antibody. Examples of serial sections through cysts that consistently either stained (A and B) or did not stain (C and D). Other lesions present in other parts of sections shown in (C and D) stained for p-S6 (data not shown), confirming that the antibody worked successfully. Bar, 100 µm.

 
Size and morphology of p-S6-stained and unstained cysts. We found no significant difference in the size of cysts that stained for p-S6 (average size, 21,441 µm2; range, 3,177-151,578 µm2) and those that did not (average, 10,615 µm2; range, 3,264-25,661 µm2), nor did we find any gross morphologic differences. In terms of cells that lined the cysts, p-S6-stained cells were of a similar size to unstained cells (100.4 versus 106.8 µm2) and these cells were of a similar size to adjacent normal tubular epithelial cells (98.9 µm2). Furthermore, cells lining both p-S6-stained and unstained cysts had similar morphologies. In contrast, cells from cystadenomas and renal cell carcinomas were significantly larger than cells from cysts or adjacent normal tubular epithelial cells (172.7 and 188.3 µm2, respectively; P < 0.001).

Analysis of renal lesions from Tsc1+/– Blm–/– mice. Normally, a diverse range of mutations, such as nonsense, frameshift, and deletion mutations and epigenetic silencing, may inactivate the wild-type alleles of tumor suppressor genes, and this diversity makes their identification problematic. We have overcome this problem by crossing our Tsc1+/– mice onto a Blm-deficient background (20), which increases the frequency of somatic LOH of the Tsc1 wild-type allele (19), thereby facilitating the detection of second hits. As observed in Tsc1+/– mice, cysts from Tsc1+/– Blm–/– mice were smaller and more numerous than cystadenomas and renal cell carcinomas, supporting the model of tumor progression. We sought somatic LOH events and intragenic mutations at the Tsc1 locus in DNA extracted from 23 renal cysts, 38 renal cystadenomas, and 24 renal cell carcinomas after laser capture microdissection from Tsc1+/– Blm–/– mice. In total, we identified 70 of 85 (82.4%) somatic Tsc1 mutations, which included 69 LOH events and 1 intragenic mutation. However, in 34.8% (8 of 23) of cysts, we did not identify any somatic Tsc1 mutations, as compared with only 13.2% (5 of 38) of cystadenomas and 8.3% (2 of 24) of renal cell carcinomas (Table 3 ). Therefore, in agreement with our studies on Tsc1+/– mice, somatic Tsc1 mutations were significantly less frequent in cysts as compared with cystadenomas (P = 0.048) and renal cell carcinomas (P = 0.03) on a Tsc1+/– Blm–/– background. We also assessed the expression levels of p-mTOR in 23 renal cysts, 42 renal cystadenomas, and 9 renal cell carcinomas from Tsc1+/– Blm–/– mice by immunohistochemistry, and, consistent with our previous findings, we observed that significantly fewer cysts had activation of the mTOR pathway as compared with the more advanced lesions (P = 0.02).


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  		Table 3. Somatic Tsc1 mutations in renal lesions from Tsc1+/– Blm–/– mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A wide range of mutational mechanisms may cause somatic inactivation of the wild-type allele of tumor suppressor genes. Consequently, reports of haploinsufficiency at tumor suppressor loci may sometimes be incorrect and simply reflect an inability to find the somatic mutations due to inadequate mutation analyses. We have used two complimentary approaches to show Tsc1 haploinsufficiency in a proportion of renal cysts from Tsc1+/– mice. First, we have shown by staining for p-mTOR and p-S6 that there is little or no activation of the mTOR pathway in ~37% to 53% of cysts. Second, we have crossed Tsc1+/– mice onto a Blm-deficient background, which dramatically increases the frequency of somatic LOH of the wild-type Tsc1 allele, making the identification of second hits straightforward (19); on this background, ~34.8% of cysts were haploinsufficient for Tsc1. Interestingly, second hits at Tsc1 and activation of the mTOR pathway were found in the vast majority of more advanced renal tumors, suggesting that although this is an important step in TSC-associated renal tumorigenesis, it may not be the key initiating event in this process.

Recent work on the TSC signal transduction pathway has led to the discovery of links between components of this pathway and genes mutated in other hamartoma syndromes (reviewed in ref. 9). Phosphatase and tensin homologue (PTEN; mutated in Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease, and Proteus syndrome) encodes a dual-specificity phosphatase that negatively regulates the PI3K/Akt pathway; LKB1/STK11 (mutated in Peutz-Jeghers syndrome) encodes a protein kinase that phosphorylates and activates the AMP-activated protein kinase (a positive regulator of the TSC1-TSC2 complex); and SMAD4 (mutated in juvenile polyposis) intereacts with SMAD3, which itself interacts with Akt and TSC2. Interestingly, hyperplastic-dysplastic colon mucosa and polyps from Pten+/– mice retain the wild-type Pten allele (21); gastrointestinal hamartomas from Lkb1+/– mice retain the wild-type Lkb1 allele (22, 23); and early gastrointestinal serrated adenomas and polyps from Smad4+/E6sad mice retain the wild-type Smad4 allele (24, 25). Furthermore, more advanced lesions from these mice generally exhibit somatic inactivation of their respective wild-type alleles (24–26). Therefore, our results suggest a possible common mechanism of tumor initiation in these hamartoma syndromes, whereby one hit initiates tumorigenesis and two hits promotes the progression to more advanced lesions.

Other studies have also suggested that haploinsufficiency for TSC1 or TSC2 has both biochemical and phenotypic consequences. Phenotypically normal renal epithelial cells from TSC mutation carriers have significant differences in gene expression profiles compared with similar cells from controls (27); Tsc1+/– and Tsc2+/– mice exhibit increased numbers of astrocytes (28); young Eker rats (that rarely harbor brain lesions) exhibit enhanced responses to chemically-induced kindling (29); and Tsc1 haploinsufficient neurons have increased soma size, decreased spine density, and increased spine length and head width (30). Furthermore, this neuronal morphology becomes more pronounced after loss of the 2nd Tsc1 allele, suggesting that the TSC pathway is sensitive to gene dosage (30). Further studies are therefore warranted to determine the effects of TSC1 and TSC2 haploinsufficiency and to define the key event(s) in tumor initiation.

It has recently been suggested that rapamycin, an mTOR inhibitor, may be an effective drug in treating patients with TSC and other mTOR activation syndromes. Studies have shown that rapamycin treatment of Eker rats resulted in a significant decrease in the size of the Tsc2-related renal tumors, accompanied by down-regulation of p-S6 activity (31). However, rapamycin had no effect on the number of microscopic precursor lesions, suggesting a rapamycin-insensitive pathway during Tsc2-associated tumor initiation (31). Our data may also indicate an mTOR-independent pathway during Tsc1-associated tumor initiation. Therefore, although rapamycin may help control TSC-associated tumor development, it may not prevent tumor initiation.


   Acknowledgments
 
Grant support: Tenovus, the Tuberous Sclerosis Association, the Tuberous Sclerosis Alliance, and the Wales Gene Park.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. D.F.R. Griffiths for helpful advice.

Received 5/12/06. Revised 6/20/06. Accepted 6/22/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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