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Transforming Growth Factor-ß Receptor Type I Gene Is Frequently Mutated in Ovarian Carcinomas¹

Wood Hudson Cancer Research Laboratory, Newport, KY 41071-4701 [T. C., J. T., B. D., B. H., B. C., J. H. C.]; Department of Pathology and Laboratory Medicine, St. Elizabeth Medical Center, Edgewood, KY 41017 [J. P.]; and Lilly Research Laboratory, Cancer Research Division, Eli Lilly and Company, Indianapolis, Indiana 46285 [J. R. G.]

	ABSTRACT

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Ovarian carcinomas (OCs), particularly recurrent OCs, are frequently resistant to transforming growth factor (TGF)-ß-mediated growth inhibition. Mutations in the TGF-ß receptor type II (TßR-II) gene are only evident in a minority of OCs, suggesting that other alterations of the TGF-ß signaling pathway may be involved in OC. Using PCR, cold single-strand conformation polymorphism, and DNA sequencing, we now show that 33% of primary OCs (10 of 30) harbor somatic changes in exons 2, 3, 4, and 6 of the TGF-ß receptor I (TßR-I) gene. Most of the changes are missense mutations and clustered largely in the catalytic domain of the receptor kinase. Interestingly, seven additional cases (23.3%) showed heterozygous carriers of an allelic variant [a 9-nucleotide deletion, del(GGC)₃] in exon 1 of the TßR-I gene. This is in contrast with 10.6% of del(GGC)₃ heterozygous carriers in a recent report of a large normal population (n = 735; B. Pasche et al., Cancer Res., 59: 5678–5682, 1999). These results indicate that TßR-I is frequently mutated in OC and suggest that resistance to TGF-ß-mediated growth inhibition may frequently involve alterations of the TßR-I gene.

	Introduction

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OC³ is a very aggressive type of cancer among women. Each year, 23,400 women in the United States will be diagnosed with OC, and more than half of them will die from the disease (1) . OCs, particularly recurrent OCs, are frequently resistant to growth inhibition by TGF-ß (2, 3, 4) , suggesting that alterations in the TGF-ß response are involved with OC progression (3) .

The TGF-ß signal is transduced by two transmembrane serine-threonine kinase receptors. TGF-ß binds first to TßR-II homodimers, which then form heterotetramers with two TßR-I molecules. As a consequence, the TßR-II kinase phosphorylates and activates TßR-I (5) . Activated TßR-I then phosphorylates Smad2 and Smad3, which in turn heterotrimerize with Smad4. These Smad complexes then translocate to the nucleus, bind to DNA in a sequence-specific manner, and regulate gene transcription, leading to cell cycle arrest (5, 6, 7, 8) .

Alterations in these key effectors of the TGF-ß signaling pathway have been identified in numerous human tumors and account for the loss of TGF-ß responsiveness (9, 10, 11, 12, 13) . For instance, TßR-II mutations are frequent in hereditary nonpolyposis colorectal cancer, and homozygous deletion of the Smad4 gene is frequent in pancreatic cancer (11 , 13) . In ovarian cancers, TGF-ß resistance is frequent, perhaps exceeding 75% of the cases (2 , 4) . Code-altering mutations in TßR-II have been reported in a minority of human OCs (14) suggesting that, in ovarian carcinogenesis, alterations in other components of TGF-ß signaling may play a role in the loss of TGF-ß responsiveness. We have shown recently that TßR-I is frequently inactivated by mutation in metastatic breast cancers and head and neck cancers (15 , 16) . We therefore sought to explore whether TßR-I mutations may be involved in ovarian carcinogenesis. Our data indicate that 33% of OCs show code-altering, somatic mutations. Moreover, our data also indicate that 23% of OCs harbor a 9-bp deletion of exon 1 that has been defined previously as a germ-line deletion (17 , 18) . Together, these data suggest that alterations in TßR-I are frequent in OCs and may be intimately involved in the frequent loss of TGF-ß responsiveness that typifies OC.

	Materials and Methods

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Clinical Specimens.
Paraffin-embedded OC tissues are archived at Wood Hudson Cancer Research Laboratory. The tissues were obtained at surgery at St. Elizabeth Medical Center (Covington/Edgewood, KY). Institutional Review Board approval for this study was received from St. Elizabeth Medical Center. A board-certified pathologist (J. P.) reviewed a H&E-stained section from each block of ovarian tissue and graded the tumors according to WHO classification and Nomenclature of Ovarian Neoplasms. We analyzed 30 cases with a diagnosis of OC including tumors of low malignant potential to grade IV. The average age of patients at diagnosis was 62.2 years.

Paraffin-embedded Tissue Microdissection.
The basic technical approach has been described previously (19) . Briefly, a single 8–10-µm-thick paraffin section was stained and manually microdissected for each tumor sample. Using companion H&E-stained slides as a reference, tumor cells were microdissected with a fine-point surgical blade (no. 11) under an inverted microscope. Adjacent normal tissue of the same individual was dissected the same way to determine the somatic or germ-line nature of genetic changes.

DNA Preparation.
Microdissected samples were deparaffinized (three washes with xylene for 30 min each) and rehydrated in decreasing concentration of alcohol (19) . DNA was extracted with Instagene Chelex matrix solution according to the manufacturer’s instructions (Bio-Rad, Hercules, CA), containing 60 µg of proteinase K in a shaking incubator at 37°C overnight. Samples were boiled for 10 min, vortexed, and centrifuged at 7000 x g for 5 min. Approximately 2–8 µl of supernatant were used for PCR amplification.

PCR-SSCP.
A sensitive "cold" PCR-SSCP was used to screen for mutations (20) . Primers used for PCR amplification of the 9 exons of the TßR-I gene were targeted to intronic sequences to amplify the complete coding sequence of each exon. Primer sequences were described previously (15) . The PCR reaction was performed in a volume of 20 µl containing 500 nM unlabeled primers and 2 units of AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Branchburg, NJ). After an initial 10-min denaturation at 95°C, PCR was run for 50 cycles of 95°C for 1 min, 55°C for 40 s, and 72°C for 1 min, followed by a 5-min final extension at 72°C. For PCR amplification of the GC-rich exon 1 of TßR-I, we added 2 M betaine in the PCR mixture. For SSCP analysis, a 5-µl aliquot of amplified PCR product was mixed with 15 µl of loading buffer (12.5 µl of 10x TBE buffer, 2 µl of 15% Ficoll, 0.1% bromphenol blue and xylene cyanol, and 0.5 µl of methyl mercury hydroxide), denatured by heating at 75°C for 3 min, and quenched on ice. The single-stranded DNA fragments were then resolved using precast 20% TBE acrylamide gels on a Novex X-cell II Thermoflow apparatus (Novex, San Diego, CA), with the gel temperature maintained precisely at 10°C throughout the run. Bands were visualized by staining the gel in a 1:10,000 dilution of SYBR Green II (Molecular Probes, Inc., Eugene, OR) for 20–30 min, and photographic documentation was carried out by using a camera coupled with a SYBR Green band pass filter.

Sequencing of the Mutant Fragments.
Suspect bands detected in cold SSCP were first verified by an additional, independent PCR-SSCP analysis. DNA from adjacent normal tissue was included in this process. The confirmed shifts in the SSCP gel were excised for sequencing. After reamplification of the shifted bands, the PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA). Finally, purified DNA fragments were directly sequenced by the same forward or reverse primers used in the original PCR amplification. The sequencing was done in an automated sequencer (PE Applied Biosystems Model 377) at the DNA Core Facility of the University of Cincinnati (Cincinnati, OH).

	Results

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Somatic Mutations of the TßR-I Gene in Ovarian Cancer.
To assess the incidence of somatic mutations in ovarian carcinoma, we have microdissected tumor from normal cells and screened all nine exons of the TßR-I gene in a total of 30 OC cases. Ten tumor cases showed confirmed bandshifts that were different from the wild-type bands among exons 2, 3, 4, and 6 of the TßR-I gene (Fig. 1) . Most of the shifts were exclusive to tumor tissues and are therefore indicative of somatic mutations. These included two shifts in exon 2, two shifts in exon 3, five shifts in exon 4, and three shifts in exon 6 (Table 1) . One case (OC18) showed a strong shift in the tumor and a weak shift in the adjacent normal stroma tissues but not in different non-tumor blocks of the same patient (Fig. 1) . Sequencing of these shifted bands revealed that each carries a point mutation, most of which were missense mutations (Fig. 2A) . There was only one silent mutation of exon 4 in case OC6 among all genetic changes found in this study (Table 1) . However, this patient harbored another missense mutation in exon 3. Mutations were not found in exons 5, 7, 8, or 9 of the TßR-I gene. Table 1 summarizes the clinical information for all patients studied and the mutations, including their locations at different exons and amino acid substitutions. There was no apparent relationship between OC subtype and TßR-I mutations (Table 1) .

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative photograph of somatic mutations detected by PCR-SSCP. All OC samples with the aberrant bands that appeared different from wild-type pattern in the SSCP gel were subject to confirmation. Both tumor (T) and adjacent normal (N) tissues were confirmed independently for the shifts. Arrows, shifted bands that are present only in tumor lanes but absent in corresponding normal lanes. Note the weak shifted band in one normal sample of OC18 in addition to the strong shift in the tumor lane.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Missense mutations in OCs. A, representative sequence chromatograms of six PCR fragments that were directly sequenced after PCR-SSCP. Five transition (CT, GA), and one transversion (CA) nucleotide changes resulted in missense mutations in exon 2 (T98I), exon 3 (V145I), exon 4 (V206M, S210N, and R225Q), and exon 6 (P327Q). B, schematic diagram of functional domains of the TßR-I gene and locations of mutations. Frequent mutations of S210N, R225Q, and P327Q are localized in the kinase domain. V206M is at the junction between GS domain and kinase domain. V145I is in the transmembrane (TM) domain. del(GGC)3 is at a hydrophobic signal sequence of the protein.

In exon 6, three cases showed the same mutation, a C-to-A transversion mutation resulting in proline-to-glutamine substitution at codon 327 (P327Q). Codon 327 is highly conserved in all type I receptor kinases (TßR-I, ActR-IB, ActRI, BMPRI-B, tkv, and TßR-II), which suggests that mutations of this codon (P327Q) may have functional consequences (21) . Moreover, this mutation sits right in the E6 loop that is critical for receptor dimerization, as described in studies of the crystal structure of TßR-I (21) .

Additional somatic mutations in the transmembrane and extracellular domains were also found in this study. Case 10 had a mutation in exon 2 that results in a threonine-to-isoleucine substitution (T98I) located in the extracellular domain. Case 6 had a mutation inside the transmembrane domain that resulted in a valine-to-isoleucine substitution (V145I). Case 21 had a mutation in exon 3, substituting histidine for tyrosine (H149Y), that might affect the cytoplasmic juxtamembrane region.

Frequent Allelic Variants of the TßR-I Gene in OC.
A 9-bp, germ-line deletion (heterozygous allelic variant) in exon 1 of TßR-I [del(GGC)₃] was reported in our previous studies of cervical cancer (17 , 18) and in a large study of the normal population (22) . To evaluate the prevalence of this allelic variant form in OCs, we amplified exon 1 of the TßR-I gene in all 30 cases of OC. Because exon 1 is located in a highly GC-rich region and is relatively difficult to amplify, we optimized the PCR amplification by including 2 M betaine in the PCR mixture. By using sensitive "cold" SSCP methods, we were able to successfully identify the deleted allele from the wild-type allele (Fig. 3) . We found that 7 of 30 (23.3%) ovarian cancer cases showed this allelic variant by SSCP. Each of the cases with del(GGC)₃ allele also retained the wild-type allele (Fig. 3) . Furthermore, using tissue microdissection, we identified this deletion in both the tumor and the adjacent stroma. In fact, the deleted allele and the wild-type allele were evident at roughly the same intensity in both tumor and stroma, suggesting that this deletion represents the germ-line deletion that had been identified previously (18) . DNA sequencing confirmed the presence of both the wild-type and variant del(GGC)₃ alleles. This deletion would eliminate three alanines in the signal peptide of the receptor. Interestingly, the frequency of the del(GGC)₃ allele in this study of OCs was 23.3%, more than twice that reported in the normal population (18 , 22) .

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Germ-line deletions of the TßR-I gene in OC detected by PCR-SSCP. Arrows, full-length and deleted forms of exon 1. Lanes 1, 5, and 9, wild type for exon 1; Lanes 2–4 and 6–8, cases with germ-line deletion of exon 1, and they are all heterozygous del(GGC)3 carriers. Lanes 7 and 8 are from right and left ovaries of the same case.

	Discussion

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The crystal structure of the cytosolic portion of TßR-I protein has been determined recently. Functionally critical segments include the GS loop, the phosphate-binding loop, and the catalytic segment (21) . In this report, we identified five missense mutations in exon 4 and three mutations in exon 6 of the TßR-I gene that alter critical codons within the TßR-I kinase domain and may disrupt receptor kinase activity. The mutations in exon 4 are located in the phosphate-binding loop adjacent to the GS domain and may have detrimental effects on activation of receptor kinase or ATP binding. The proline-to-glutamine substitution (P327Q) in exon 6 occurred in three cases. This residue is highly conserved in all type I receptor kinases and sits within the E6 loop that is critical for receptor dimerization (21) . Mutations of this codon may therefore affect receptor dimerization. We have also identified mutations in exons 2 and 3, which are located in the extracellular domain and transmembrane domain, respectively. These mutations may affect ligand binding capability during formation of the ligand/receptor complex. Importantly, the mutations identified in this study were not the same as those identified in our previous studies of breast and head and neck carcinomas, suggesting that tissue-specific factors might influence the accumulation of genetic changes in different cancer types.

Recently, Wang et al. (23) also reported somatic mutations in TßR-I in 31% of primary ovarian carcinomas. However, these were defined as frameshift mutations exclusively in exon 5 of TßR-I. In this report, we did not find any mutations in exon 5. It seems plausible that these differences may reflect differences in the patient populations examined. Nevertheless, both studies indicate that somatic alterations in TßR-I are frequent in human OC, further strengthening the notion that these alterations may be related to the loss of TGF-ß responsiveness.

In addition to the somatic mutations, we have also shown a high frequency of allelic variant in exon 1 [germ-line del(GGC)₃]. This deletion has been defined previously as a germ-line mutation in a large study of the normal population and in our study of cervical cancer (18 , 22) . In this study, 7 of 30 (23.3%) OC cases showed the allelic variant of exon 1. This deletion was evident in the tumor and adjacent stroma, consistent with a germ-line nature. Moreover, the frequency of this deletion is more than twice that found in the normal population (10.6%) in a recent study (22) . In the clinical follow-up analysis, two of the patients (OC1 and OC15) with the deletion are among the younger ages in the group analyzed (Table 1) . The average ages of the patients with del(GGC)₃ variant was 57.1 but was 63.8 for the rest of patients. Interestingly, six of seven cases with this allelic variant had poorly differentiated or undifferentiated carcinomas (grade 3 or 4; see Table 1 ). Together these data suggest that this deletion may contribute to, or predispose individuals to, OC. However, a large case-control study will be needed to further explore the association of this allelic variant with clinical outcome.

In each of the cases showing genetic mutations of TßR-I, both the mutant and wild-type alleles are retained (Figs. 1 and 3 ). It is plausible that haploinsufficiency for a fully functional TßR-1 may contribute to OC. Alternatively, these mutant alleles may act as dominant negatives, disrupting the function of the heterotetrameric TßR-II and TßR-I complexes. It is also possible that expression of the wild-type allele is eliminated by methylation or epigenetic inactivation (24) . Additional studies will help dissect the precise role for these TßR-I mutations in the development and progression of ovarian carcinogenesis.

In summary, this report demonstrates that code-altering mutations of TßR-I, as well as a high frequency of a germ-line deletion, are frequent in human OC. The majority of somatic mutations are clustered in critically functional domains of the receptor kinase, suggesting that selection of the genetic changes might be functionally beneficial in OC development. Together, these data indicate that the loss of TGF-ß responsiveness that typifies ovarian carcinogenesis and progression may frequently involve alterations in the TßR-I gene.

	ACKNOWLEDGMENTS

 
We thank Dr. Eric Hugo for computational assistance and many helpful suggestions for the project.

	FOOTNOTES

 
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.

¹ Supported in part by Eli Lilly & Company and The William Randolph Hearst Foundation.

² To whom requests for reprints should be addressed, at Wood Hudson Cancer Research Laboratory, 931 Isabella Street, Newport, KY 41071-4701. Phone: (859) 581-7249; Fax: (859) 581-2392; E-mail: tchen{at}woodhudson.org

³ The abbreviations used are: OC, ovarian carcinoma; TGF, transforming growth factor; TßR-I and -II, TGF-ß receptor types I and II; SSCP, single-strand conformation polymorphism.

Received 2/12/01. Accepted 4/30/01.

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