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Int 7 G 24 A Variant of Transforming Growth Factor-B Receptor Type I Is Associatedwith Invasive Breast Cancer
| Content Provider | Semantic Scholar |
|---|---|
| Author | Chen, Taiping Jackson, Chad R. Link, Andrew J. Markey, Michael P. Colligan, Bruce M. Douglass, Larry E. Pemberton, Jackson O. Deddens, James A. Graff, Jeremy R. Carter, Hannah |
| Copyright Year | 2006 |
| Abstract | Purpose: The transforming growth factor-h (TGF-h) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) ofTGF-h receptor type I (TGFBR1) is associatedwith kidney andbladder cancers in our recent study.We hypothesize that this germline variant may be involved in development and progression of breast cancer. Experimental Design: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic areawith a homogenous ethnic population.We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and153 noncancer controls.The Int7G24Awas identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in theTGFBR1were also analyzed by PCRand single-strand conformational polymorphism. Results: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2%) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutationwas found in 71breast cancers. Conclusion:These data suggest that the germline Int7G24A variant may represent a risk factor for invasivebreastcancerandamarker forbreastcancerprogression.A separate studywithalarger sample size is warranted to validate the association of the Int7G24Awithhumanbreast cancer. Breast cancer is the most common form of malignancy among women in the United States with 211,240 new cases and 40,410 deaths from the disease anticipated in 2005 (1). High-penetrance germline mutations in BRCA1 and BRCA2 have been linked to familial breast cancer susceptibility (2, 3). Polymorphisms or genetic variations in low-penetrance genes are suspected to play a role in development and progression of sporadic breast cancers (4, 5). We have now found a genetic variant in the transforming growth factor-h (TGF-h) receptor type I (TGFBR1) gene that is significantly associated with patients having invasive and metastatic breast cancers. TGF-h is a pleiotropic growth factor expressed by many cell lines and tissue types (6–9). Several important biological events are governed by this growth factor, such as cell growth, tissue differentiation, production and degradation of extracellular matrix, morphogenesis, and apoptosis (10–16). The TGFh signal is transduced by a membrane-bound serine/threonine kinase receptor complex, including TGF-h type I and II receptors. Activated TGFBR1 phosphorylates intermediates, such as SMAD2 and SMAD3, which in turn heterotrimerize with SMAD4. These SMAD complexes translocate to the nucleus, bind to DNA in a sequence-specific manner, and regulate the transcription of target genes involved in many cellular functions (17). TGFBR1 has a rate-limiting role in the signaling pathway. Any quantitative and qualitative changes in TGFBR1 will be expected to affect TGF-h-mediated growth inhibition in normal epithelial cells. Tumor cells frequently lose responsiveness to TGF-h-mediated growth inhibition due to disruption in the TGF-h signaling pathway (18–20). Increased expression of TGF-h associated with advanced stages of human breast cancer and poor prognosis are usually the outcomes for these patients (21–24). Somatic and germline mutations in the components of the TGF-h signaling pathway have been suspected to play a role in human cancer development and progression. While failing to find somatic mutations of the TGFBR1 gene in breast cancer, we did find a strong association between a germline variant in TGFBR1 and the patients having advanced breast cancer. This finding suggests that this germline variant may have the potential to be an important genetic marker for susceptibility to invasive breast cancer. Human Cancer Biology Authors’ Affiliations: Wood Hudson Cancer Research Laboratory, Newport, Kentucky; Department of Pathology and Laboratory Medicine, St. Elizabeth Medical Center, Edgewood, Kentucky; Department of Mathematical Sciences, University of Cincinnati, Cincinnati, Ohio; and Lilly Research Laboratory, Cancer Cell Growth & Translational Genetics, Eli Lilly & Co., Indianapolis, Indiana Received 7/12/05; revised10/21/05; accepted11/1/05. Grant support: Milheim Foundation,William Randolph Hearst Foundation, and Wood Hudson Cancer Research Laboratory Memorial Fund. 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 with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints:Taiping Chen,WoodHudson Cancer Research Laboratory, 931 Isabella Street, Newport, KY 41071-4701. Phone: 859-581-7249; Fax: 859581-2392; E-mail: tchen@woodhudson.org. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1518 www.aacrjournals.org Clin Cancer Res 2006;12(2) January15, 2006 392 Cancer Research. on September 6, 2017. © 2006 American Association for clincancerres.aacrjournals.org Downloaded from Materials and Methods Subjects. Archived paraffin-embedded surgical specimens from patients (diagnosed during 1985-1995) with breast cancer and noncancer controls were selected from the tissue bank at Wood Hudson Cancer Research Laboratory. Tissues were obtained at surgery at St. Elizabeth Medical Center (Covington/Edgewood, KY). Both cancer and control patients were from the same geographic area. Institutional review board approval for this study was received from St. Elizabeth Medical Center. Board-certified pathologists (L.D. and J.P.) reviewed a H&E-stained section from each block of breast tissue to confirm the histopathology. We analyzed 223 cases with a diagnosis of ductal carcinoma in situ (DCIS), infiltrating ductal carcinoma (IDC)/infiltrating lobular carcinoma (ILC), and metastatic breast cancer and 153 noncancer female controls. The ethnic background and age of each cancer patient was determined from the Kentucky Tumor Registry. Among the 223 patients with breast cancer studied, 2 (0.89%) were African American and 221 (99.1%) patients were Caucasians. The average age of cancer patients at diagnosis was 62 F 13 years (median age, 65 years; range, 34-88 years). Nontumor control specimens were obtained from excess tissues removed during surgery from patients without a diagnosis of cancer (mean age, 62 F 12.4 years; median age, 62 years; range, 23-92 years). Information on ethnic status was not available from the pathology reports accompanying the specimens. However, the 1990 U.S. Census figures for the geographic area served by St. Elizabeth Medical Center show that 97% are Caucasian, <2% are African American, and 1% of population are from other ethnic backgrounds. Most of the control specimens were tonsils, hernia sac, shavings, appendix, and noncancer biopsies from different organs. Isolation of DNA from paraffin-embedded tissues. A single 8to 10Am-thick paraffin section from each sample block was deparaffinized (three washes with xylene for 30 minutes each) and rehydrated in decreasing concentrations of alcohol (25). For identification of the Int7G24A and TGFBR1*6A alleles of TGFBR1 in cancer patients, a block without tumor was picked to extract constitutive DNA. For somatic mutation analysis, we selected blocks with tumor and used tissue microdissection to obtain tumor tissues for DNA extraction as described previously (25). DNA was extracted with Instagene chelex matrix solution containing 60 Ag proteinase K in a shaking incubator at 37jC overnight according to the manufacturer’s instructions (Bio-Rad, Hercules, CA). Samples were boiled for 10 minutes, vortexed, and centrifuged at 7,000 g for 5 minutes. The supernatant (2-8 AL) was used for PCR amplification. Annealing control primer-PCR-RFLP. An annealing control primer system was designed to improve specificity of PCR amplification (26). The unique linker of five inosines was included in the primer design. The sequences of annealing control primer primers for Int7G24A allele in the TGFBR1 gene were as follows: forward primer 5V-GTCTACCAGGCATTCGCTTCATIIIIIGCTTAGTATTCTG-3V and reverse primer 5VGCTTGACTACGATACTGTGCGAIIIIICAATTCTTGAACA-3V. The PCR was done in a volume of 20 AL containing 500 nmol/L forward and reverse primers and 2 units HotStarTaq DNA polymerase (Qiagen, Valencia, CA). After an initial 15-minute denaturation at 95jC, PCR was conducted in two stages: 5 cycles of 94jC for 1 minute, 50jC for 1 minute, 72jC for 3 minutes followed by 35 cycles of 94jC for 1 minute, 65jC for 1 minute, 72jC for 1 minute with the final extension at 72jC for 5 minutes. We used BsrI digestion of the annealing control primerPCR-amplified intron 7 fragment for identification of the Int7G24A allele. PCR (20 AL) was added with 2 AL of 10 digestion buffer containing 2.5 units BsrI. After 1.5-hour incubation at 65jC, the mixture was loaded on a 7% acrylamide gel to resolve the bands from the BsrI digestion to obtain the allelic status. Negative (H2O) and positive [known wild-type (WT) and variant carriers] controls were included throughout the experiment for quality control. The identified variant carriers had all been repeated at least twice for accuracy. PCR and single-strand conformational polymorphism. A total of 71 breast cancers were first screened for somatic mutations in the TGFBR1 gene by a ‘‘cold’’ PCR-single-strand conformational polymorphism (SSCP; ref. 27). Primer sequences for PCR amplification of the nine exons of the TGFBR1 gene were described previously (18). The PCR was done in a volume of 20 AL containing 500 nmol/L forward and reverse primers and 2 units HotStarTaq DNA polymerase. After an initial 15minute denaturation at 95jC, PCR was run for 40 cycles of 94jC for 30 seconds, 55jC for 40 seconds, 72jC for |
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| Alternate Webpage(s) | http://clincancerres.aacrjournals.org/content/clincanres/12/2/392.full.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
