Proc. Natl. Acad. Sci. USA Vol. 91, pp. 11413-11416, November 1994 Medical Sciences Predisposition to renal carcinoma in the Eker rat is determined by germ-line mutation of the tuberous sclerosis 2 (TSC2) (hereditary cancer/animal model/linkage analysis/tumor suppressor gene) RAYMOND S. YEUNG*t, GUANG-HuI XIAO*, FANG JIN*, WEN-CHING LEE*, JOSEPH R. TESTA*, AND ALFRED G. KNUDSON: Divisions of *Medical Science and *Basic Science, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 Contributed by Alfred G. Knudson, August 22, 1994

ABSTRACT Genetic predisposition to neoplasia often in- define the extent of syntenic conservation between RNO10 volves tumor suppressor . One such model of hereditary and HSA16, DNA probes from the region HSA16pl3.13-pter renal carcinoma was described in the rat by Eker. These were mapped in the rat, revealing an extended region of tumors share morphologic similarities with human renal can- synteny (R.S.Y., unpublished data). Using a combined po- cer. Linkage analysis localized the inherited mutation to rat sitional cloning and candidate gene approach, we have iden- band 10q12. This region is syntenic with human tified the Eker mutation as a rearrangement in rTsc2, the rat chromosome band 16p13.3, the site of the tuberous sclerosis 2 homologue of a cloned human tumor suppressor gene, tu- (TSC2) gene. A specific rearrangement of the rat homologue of berous sclerosis 2 (TSC2), that maps to HSA16p13.3 (10). TSC2 was found to cosegregate with carriers of the predispos- ing mutation. Tumors with or without loss of heterozygosity MATERIALS AND METHODS expressed only the mutant allele, consistent with the two-hit hypothesis. This mutation gave rise to an aberrant transcript Linkage Analysis. Genetic mapping ofinformative markers that deletes the 3' end normally containing a region of homol- utilized 100 backcross progeny generated through matings ogy with the catalytic domain of raplGAP. between the Eker male carriers and two sets of female rats from genealogically unrelated strains, Brown Norway and Fisher, as described (6). Genomic DNA was extracted from Hereditary cancers serve as models for studying the role of livers by using proteinase K/phenol procedures, and South- tumor suppressor genes in carcinogenesis. The pathogenesis ern blot analysis was performed by a standard protocol (11). of these disorders tends to follow the classic paradigm of the DNA probes were labeled with 32p using a random primer kit two-hit hypothesis described for retinoblastoma (1). Of the (Stratagene). RNO10 loci were ordered by multipoint likeli- dozen or so cloned genes that are responsible for human hood calculations performed with the CRIMAP program as hereditary cancer syndromes, all but three share features of described (6). tumor suppressor genes, exceptions being the RET protoon- Cell Lines. Independent primary renal tumors were sus- cogene in MEN2/familial medullary thyroid carcinoma syn- pended in serum-free hormone-supplemented medium and drome and the DNA mismatch repair genes (hMLHI, propagated as epithelial tumor cell lines as described (6). hMSH2, where h is human) in hereditary nonpolyposis colon Cells were harvested for DNA and RNA analyses. cancer syndrome. Based on the number of unique human PCR. The human probe for TSC2 was generated by PCR cancers that exist in hereditary forms, it has been estimated based on the published cDNA sequence (10). The primers that 50 or so tumor suppressor genes may be important in were as follows: HTS2F, 5'-GAAGGTGCTGAAGCTG- tumorigenesis. Some of these have been mapped but not yet GTTC-3'; HTS1R, 5'-CTGGCTGTGGCTGAGATGT-3'. An cloned. In addition there is one animal model that has aliquot ofa human kidney cDNA library was used as template appeared to point to yet another such gene, namely, hered- and amplified using the following conditions: 94°C for 3 min, itary renal carcinoma (RC) in the rat, originally described by 92°C for 1 min, 58°C for 1 min, and 72°C for 1 min for 30 Eker and coworkers (2, 3). cycles, followed by a 7-min extension at 72°C. The 405-bp Evidence that tumor suppressor genes are involved in the PCR product was cloned into the pGEM-T vector (Promega) Eker rat included the presence of nonrandom chromosomal and used as a probe for subsequent screening procedures. alterations (4, 5) and loss of heterozygosity (6) in the tumors. Genomic Clones. Phage clones of rTsc2 were screened Furthermore, microcell transfer of human chromosome using the 405-bp human PCR product from a Wistar rat spleen (HSA) 3 into an Eker-derived tumor cell line partially sup- genomic library (Stratagene). Inserts were subcloned into pressed growth (7). This finding was of particular interest pBluescript (SK-) (Stratagene). because HSA3p is the site of the von Hippel-Lindau syn- RNA Isolation, Northern Blot Analysis, and cDNA Library. drome gene, which predisposes to RC in humans. However, Various tissues were excised from wild-type Fisher and Eker our studies failed to find linkage between the Eker locus and rats and frozen immediately in liquid nitrogen. Frozen tissues homologous genes on HSA 3p (5). We then localized the Eker were pulverized under liquid nitrogen, resuspended in lysis gene by linkage analysis to rat chromosome (RNO) 10q12 (6); buffer [4 M guanidinium isothiocyanate/50 mM Tris HCI, pH Hino et al. (8) have confirmed this localization. This finding 7.6/10 mM EDTA/2% (wt/vol) N-laurylsarcosine], and ho- eliminated the von Hippel-Lindau locus as the inherited mogenized with a Polytron homogenizer. Total RNA was mutation, since it was mapped to RNO4q42 (6). The gene isolated by the method of Chomczynski and Sacchi (12). protamine 1 (Prml), located in the proximal third of RNO10, mRNA was purified from total RNA using oligo(dT)-celiulose was within 5 centimorgans of the RC locus and its human spin columns (Pharmacia). RNA was separated in denaturing homologue has been mapped to 16p13.13 (9). To further 0.8% agarose/formaldehyde gels, transferred to Hybond-N+

The publication costs of this article were defrayed in part by page charge Abbreviations: RC, renal carcinoma; HSA, human chromosome; payment. This article must therefore be hereby marked "advertisement" RNO, rat chromosome. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

11413 Downloaded by guest on September 26, 2021 11414 Medical Sciences: Yeung et al. Proc. Natl. Acad. Sci. USA 91 (1994) membrane (Amersham), hybridized, and washed (20). Uni- tumors from patients with tuberous sclerosis, suggesting that directional cDNA libraries were constructed from normal it may function as a tumor suppressor gene. To test this kidneys of a 1-month-old Wistar rat using a AZAP cDNA hypothesis, a 405-bp human cDNA probe was generated by synthesis kit (Stratagene). To avoid the 3' bias in the libraries, PCR amplification from a human kidney cDNA library, by both oligo(dT) and random primers were employed in the using primers based on the published cDNA sequence (10). first-strand cDNA synthesis. Approximately 2 x 106 clones Its authenticity was confirmed by a hemi-nested PCR, re- were obtained for each library. Screening was performed striction mapping, and sequencing. This single copy probe with duplicate filters using standard procedures after the was used to screen a rat genomic phage library, and four libraries had been amplified once to a titer of 1010 plaque- positive clones were recovered. A 1.6-kb EcoPJ subclone forming units/ml. Positive clones were rescued in pBlue- was used to identify the presence of restriction script (SK-) (Stratagene) and the inserts were sequenced fragment using Sequenase T7 DNA polymerase (United States Bio- length polymorphisms among the backcross strains but none chemical) with dideoxynucleotide terminators. Sequences was found. However, a rearranged BamHI fragment was were compared using the GCG sequence analysis software observed in a backcross carrier. Further analysis revealed package (Genetics Computer Group, Madison, WI). rearrangement in 5 of 14 restriction enzyme digests of ge- Fluorescence in Siu Hybridization. A rTsc2 genomic phage nomic DNA specific to the affected progeny (Fig. 1). In the clone, 9-1, was labeled with biotin-11-dUTP by nick trans- backcross analysis, the aberrant fragments were found to lation (Oncor). Fifty nanograms of DNA was annealed with cosegregate with the phenotype in all 100 F2 offspring except a 50-fold excess of total rat DNA and then hybridized to rat for one. The discordant animal was the same as that seen with embryonic fibroblast metaphase spreads as described (13). the other two candidate probes. This suggested that the Eker Wash conditions included 50%o (vol/vol) formamide/2x SSC rat mutation involves the rTsc2 gene. at 420C followed by 0.5x SSC rinses at 370C. To confirm the physical location of rTsc2, a biotin-labeled were counterstained with diamidino-2-phenylindole and pro- phage clone was used for fluorescence in situ hybridization. pidium iodide. Images were captured with a charge-coupled Under conditions of suppression hybridization, discrete flu- device camera and merged on a computer workstation. orescent signals were visualized at RNO1Oq12 where the Eker gene is expected to map. Independently, a somatic hybrid panel segregating for all 20 rat autosomes provided RESULTS additional evidence of rTsc2 mapping to RNO10 (R.S.Y., Refining Linkage at RN010q12. Although RN010 shares unpublished data). extensive homology with HSA17, the proximal third of When loss of heterozygosity was examined in primary RNO10 is syntenic with HSA16 (6). We have found DNA loci tumors and cell lines, the aberrant rTsc2 fragment was always lying on either side of PRMI in the region of HSA16p13 to retained while the wild-type allele was lost in 21 of the 29 map to RNO1Oq11-.q12 (R.S.Y., unpublished data). These (72.4%) samples. Thus the majority of tumors contain only included the genes MRP (multidrug resistance-associated the mutant gene, consistent with the two-hit hypothesis. We ), MYHJ1 (myosin heavy chain 11), GSPT1 (Gl-to-S presume that the tumors that did not show loss of heterozy- phase transition 1), TNP2 (transition protein 2), NOP3 (single gosity had sustained subtle intragenic mutations. copy probe of D16S237), IL9R (interleukin 9 receptor), and MPG (N-methylpurine-DNA glycosylase). Five of these BC seven loci showed informative restriction fragment length F W + polymorphisms among four rat strains [Long Evans (LE), Brown Norway (BN), Wistar, and Fisher (F)]. Linkage analysis based on 100 F2 backcross progeny [40 (LE x BN) x BN and 60 (LE x F) x F] confirmed close linkage to the RC phenotype. The order of these loci with respect to the # Eker gene, based on multipoint analysis, is cent.-Myhll- A:B.... Gsptl-Prml/Tnp2-RC-Mpg/119r. The closest markers, Mpg Nhei and I19r, had one recombinant among 100 animals tested. This discordant animal was derived from the Fisher back- .t ! ground in which a small solitary renal tumor was found in one kidney but genotyping showed the absence of the Wistar allele expected in a carrier. Since spontaneous renal tumors develop in normal Fisher rats at a reported incidence as high as :0.5% (14), this represents a plausible explanation for the source of tumor in this rat. Having reached the limit of resolution in our backcross analysis, we embarked on a search for candidate genes in the region of HSA16p13.3, where MPG has been mapped (15). Candidate Genes. Mpg and l19r were considered as candi- Xbal dates for the RC locus because of their tight linkage to the Eker phenotype and because of their possible relevance to carcinogenesis. However, neither probe showed structural rearrangement by Southern blot analysis of the RC/+ car- riers and tumors. Furthermore, Northern blot analysis failed to demonstrate I19rexpression in normal rat kidneys (1 month old and we began to examine yet another adult). Therefore, FIG. 1. Examples of genomic rearrangements on Southern blot candidate gene, the tuberous sclerosis 2 (TSC2) gene that had hybridization using a 1.6-kb genomic DNA probe of rTsc2. Arrows been mapped to the telomeric region of HSA16p (10). This indicate abnormal bands specific to the Eker gene carrier. Genomic gene appeared to be a good candidate based on (i) its map DNA from livers ofnormal rats (F, Fisher; W, Wistar) and backcross location in humans, (ii) its predisposition to RC in heterozy- progeny (BC) (+, carrier; -, noncarrier) were digested with Nhe I gous carriers, and (iii) evidence of loss of heterozygosity in and Xba I (as indicated) and resolved on a 0.7% agarose gel. Downloaded by guest on September 26, 2021 Medical Sciences: Yeung et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11415 Expression ofrTsc2. A cDNA iibrary was constructed from of the 1.55-kb genomic fragment that detected the rearrange- a 1-month-old Wistar rat kidney, a period during which the ment aligned with the human sequence at positions =e3620 to target gene remains susceptible to radiation and chemical 3830with =85% identity. Based on human sequence analysis, carcinogenic effects and is, thus, presumably expressed (5, this site lies proximal to the raplGAP-related domain (10). 16). With the use of a 405-bp human cDNA probe, a 2.9-kb Partial sequencing of the rTsc2 cDNA also revealed the rat cDNA clone was isolated. When this clone was hybrid- conserved GAP-like domain near the 3' terminus with an ized to Southern blots, the same pattern of rearrangements identical predicted amino acid sequence except for a conser- that was observed with genomic probes was seen. The vative isoleucine (rat) to valine (human) substitution at po- pattern of expression in normal rat tissue identifies a single sition 1626 of the human protein sequence. A cDNA probe 3' transcript of =5.5 kb, variably expressed in a wide spectrum to the breakpoint containing this domain failed to hybridize of tissues including kidney (1 month old and adult), brain, to tumor cell line mRNA on Northern blot analysis (results heart, lung, muscle, liver, and spleen (Fig. 2 A and B). This are identical to that shown in Fig. 2D). This suggested that is consistent with the effect of the Eker mutation on various -30% of the 3' portion of the transcript is deleted by the organ tissues. The level ofexpression showed a 10- to 20-fold rearrangement. variation, with the highest in brain and lowest in liver. Expression in the kidney was approximately equal in the 1-month-old and adult animals. DISCUSSION Northern blot analysis of three renal tumor cell lines We used a combination of positional cloning and candidate showed an abnormal rTsc2 transcript of =7.5 kb in the gene approaches to identify the gene that is mutated in a absence of the normal mRNA (Fig. 2C). This suggested that dominantly heritable RC model in the rat. Our search was the mutation resulted in a rearrangement within an open guided by the finding that synteny extends between reading frame that produced a larger than normal transcript. HSA16p13.13 to -16p13.3 and the proximal third of RNO10 The 3' end ofthe normal rTsc2 mRNA was not present in the where the Eker locus was mapped (6). In particular, DNA mutant transcript (Fig. 2D). Correlation with Southern blot markers near the telomere of HSA16p showed tight linkage analysis showed that tumor cell lines 2 and 5 retained only the with the Eker phenotype, thus TSC2 became a logical can- abnormal allele, but cell line 4 was one of the eight tumors didate gene since patients with tuberous sclerosis are pre- that showed no loss of heterozygosity (Fig. 2E); we presume disposed to multiple hamartomas and neoplasia, including that a subtle mutation in the "normal" allele leads to its RC. Our findings ofa structural rearrangement specific to the inactivation. rTsc2 gene in the Eker carriers and tumors with aberrant The combination of genomic rearrangement specific to expression confirm it being the inherited mutation. Compar- carriers and tumors and the presence of tumor-specific ab- ative mapping data would also suggest the location of Tsc2 in errant transcript confirms that rTsc2 is the inherited mutation the mouse to be on chromosome 11 where a tightly linked in the Eker rat. These findings also provide strong evidence marker, Mpg, has been mapped (17). At least one other locus, that rTsc2 functions as a classic tumor suppressor gene. HBA, maps to the corresponding regions in the human and Analysis of an adult kidney derived from a histologically mouse (18). proven heterozygous carrier showed that both the normal and In the rat, germ-line mutation of rTsc2 results in a strong mutant alleles were transcribed (Fig. 2B). The level of predisposition to tumor formation, mostly involving mesen- expression of the mutant mRNA appeared lower than that of chymal-derived tissues such as the renal tubules, spleen, and the normal transcript, suggesting a possibly less stable mu- uterus. The mutation in the Eker rat involved the 3' portion tant mRNA as a result ofthe 3' extension. The primary tumor of the gene proximal to the putative raplGAP "catalytic" taken from this animal expressed only the larger transcript. domain (19). The rearrangement resulted in a new 3' terminus Nature of the Eker Mutation. The approximate location of ofthe transcript that effectively eliminates the distal 309o of the breakpoint within the normal cDNA was deduced by the mRNA containing the region of raplGAP homology. comparing the sequences of the genomic junction fragment Whether such a mechanism leads to altered function of with the human cDNA. A coding region within the 5' portion downstream effector(s) of the GTPase family involved in

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FIG. 2. (A) Northern blot analysis showing expression of rTsc2 in all of the tissues tested. Twenty micrograms of total RNA was loaded per lane; the ethidium bromide-stained gel is shown below the autoradiograph. A 2.9-kb cDNA was used as probe. CNS, central nervous system. (B) Normal and abnormal transcripts are expressed in normal kidney derived from a heterozygous carrier. Primary tumor from this animal expressed only the abnormal transcript. (C) Northern blot analysis of Eker-derived renal tumor cell lines (lanes 2, 4, and 5) and normal kidney (lane N) showed a single larger transcript in the tumors compared to normal. One microgram of mRNA was added to each lane. (D) When the 0.55-kb 3' end of the normal rTsc2 cDNA was hybridized to the same blot shown in C, no signal was detected despite prolonged exposure, indicating that the mutation disrupted the 3' terminus of the gene. (E) Southern blot analysis of corresponding tumor cell lines (lanes 4 and 5) illustrates the retention of heterozygosity in cell line 4 although no normal transcript can be detected, suggesting a subtle "second" mutation in the wild-type allele resulting in loss of expression. Lane N contains normal kidney DNA. Downloaded by guest on September 26, 2021 11416 Medical Sciences: Yeung et al. Proc. Nati. Acad. Sci. USA 91 (1994) signaling pathways regulating cell growth and differentiation 4. Funaki, K., Everitt, J., Oshimura, M., Freed, J. J., Knudson, remains to be tested. Experimental data have yet to demon- A. G. & Walker, C. (1991) Cancer Res. 51, 4415-4422. strate GAP-like activity in the normal protein. The expres- 5. Hino, O., Klein-Szanto, A. J. P., Freed, J. J., Testa, J. R., sion patterns of TSC2 in normal rat and human tissues are Brown, D. Q., Vilensky, M., Yeung, R. S., Tartof, K. D. & with the multiorgan involve- Knudson, A. G. (1993) Proc. Nat!. Acad. Sci. USA 90, 327- similarly widespread, consistent 331. ment when the gene has been mutated. 6. Yeung, R. S., Buetow, K. H., Testa, J. R. & Knudson, A. G. The molecular effects of the Eker mutation provide strong (1993) Proc. Nat!. Acad. Sci. USA 90, 8038-8042. evidence that rTsc2 behaves as a tumor suppressor gene. 7. Hiratsuka, M., Shimizu, M., Funaki, K. & Oshimura, M. (1991) Normal kidney of the heterozygous animal expressed both Proc. Jpn. Cancer Assoc. 50, 120 (abstr.). normal and abnormal rTsc2 mRNA, but primary tumor and 8. Hino, O., Mitani, H., Nishizawa, M., Katsuyama, H, Koba- cell lines exhibited only the mutant transcript. Even in the yashi, E. & Hirayama, Y. (1993) Jpn. J. Cancer Res. 84, tumor that retained heterozygosity at the rTsc2 locus, only 1106-1109. the aberrant transcript was expressed. This supports the 9. Reeders, S. T., Hildebrand, C. E. & Sutherland, G. R. (1991) notion that both copies ofthe normal gene must be disrupted Cytogenet. Cell Genet. 58, 643. before tumorigenesis ensues. Similarly, in humans, various 10. The European Tuberous Sclerosis Consortium in patients with (1993) Cell 75, 1305-1315. deletional mutations have been identified 11. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular tuberous sclerosis, and affected individuals have reduced Cloning: A Laboratory Manual (Cold Spring Harbor Lab. expression of TSC2, suggesting that the mutations are also Press, Plainview, NY), 2nd Ed., pp. 9.16-9.19. inactivating (10). 12. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. We gratefully acknowledge J. J. Freed and K. H. Buetow for 13. Yeung, R. S., Taguchi, T., Patriotis, C., Makris, A., Tsichlis, assistance and discussion related to tumor cell line propagation and P. N., Levan, K. K., Levan, G., Tartof, K., Hino, O., Knud- linkage analysis. We thank D. D. Halley and the European Chro- son, A. G. & Testa, J. R. (1993) Cytogenet. Cell Genet. 62, mosome 16 Tuberous Sclerosis Consortium for providing probes for 149-152. hTSC2, A. Stewart for hMRP, M. Siciliano for hMYHII, F. Hanaoka 14. Dietrich, D. R. & Swenberg, J. A. (1991) Mutat. Res. 248, for hGSPTI, G. Schluter for rTnp2, M. Breuning for NOP3, P. 239-260. Tsichlis for rll9r, and B. Engelward for mMpg. We further acknowl- 15. Samson, L., Derfler, B., Boosalis, M. & Call, K. (1991) Proc. edge A. Lerro and S. Horward for technical assistance. This work Natl. Acad. Sci. USA 88, 9127-9131. was supported in part by National Institutes of Health Grants 16. Walker, C., Goldsworthy, T. L., Wolf, D. C. & Everitt, J. CA61889 (R.S.Y.), CA45745 (J.R.T.), CA43211 (A.G.K.), grants (1992) Science 255, 1693-1695. from The W. W. Smith Charitable Trust, The Lucille P. Markey 17. Engelward, B. P., Boosalis, M. S., Chen, B. J., Deng, Z., Charitable Trust, the American Cancer Society-Institutional Re- Siciliano, M. J. & Samson, L. D. (1993) Carcinogenesis 14, search Grant, U.S. Public Health Service Core Grant CA06927, and 175-181. an appropriation from the Commonwealth of Pennsylvania. W-C.L. 18. O'Brien, S. J., Peters, J., Searle, A., Womack, J. & Marshall- is a fellow of the Leukemia Society of America. R.S.Y. is a Council Graves, J. (1993) in Chromosome Coordinating Meeting (1992): for Tobacco Research Scholar and recipient ofthe American Society Genome Priority Reports, eds. Cuticchia, A. J., Pearson, P. L. of Clinical Oncology Young Investigator Award. & Klinger, H. P. (Karger, Basel), Vol. 1, pp. 758-809. 19. Rubinfeld, B., Crosier, W. J., Albert, I., Conroy, L., Clark, R., 1. Knudson, A. G. (1993) Proc. Nat!. Acad. Sci. USA 90, 10914- McCormick, F. & Polakis, P. (1992) Mol. Cell. Biol. 12, 10921. 4634-4642. 2. Eker, R. (1954) Acta Pathol. Microbiol. Scand. 34, 554-562. 20. Sambrook, J, Fritsch, E. F. & Maniatis, T. (1989) Molecular 3. Eker, R., Mossige, J., Johannessen, J. V. & Aars, H. (1981) Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Diag. Histopathol. 4, 99-110. Press, Plainview, NY), 2nd Ed., pp. 7.43-7.50. Downloaded by guest on September 26, 2021