ANTICANCER RESEARCH 30: 3093-3098 (2010) CCND1 and CDKN1B Polymorphisms and Risk of Breast Cancer EMEL CANBAY1, ILHAN YAYLIM ERALTAN2, ALI CERCEL3, TURGAY ISBIR2, ERTUGRUL GAZIOGLU3, FATIH AYDOGAN3, CANAN CACINA2, ALI CENGIZ3, MEHMET FERAHMAN3, EMEL ZENGIN4 and HILAL UNAL3 1General Surgery, Basaksehir State Hospital, Istanbul 34230, Turkey; 2Department of Molecular Medicine, Experimental Medicine and Research Institute (DETAE), Istanbul University, Istanbul, 34100, Turkey; Departments of 3General Surgery and 4Biochemistry, Cerrahpasa Medical School, Istanbul University, Istanbul, 34098, Turkey Abstract. Background and Objectives. Previous studies have Breast cancer includes a heterogeneous group of tumours shown alterations in the cell cycle regulatory proteins in breast with variable prognosis and is the second leading cause of carcinomas. However, the results of these studies remain cancer related death among women (1). The genetic material controversial. Cyclin D1 (CCND1) and p27KIP1 (CDKN1B) which modifies the expression of proteins in cell cycle are two essential regulators of cell cycle progression. This progression is altered in tumourigenesis (2). The cell cycle study aimed to investigate the associations of CCND1 A870G is promoted by activation of cyclin dependent kinases, which and CDKN1B C79T polymorphisms with breast cancer risk. are positively regulated by cyclins and negatively by cyclin Patients and Methods. Polymerase chain reaction-restriction dependent kinase inhibitors. fragment length polymorphism (PCR-RFLP) was used to Cyclin D1 (CCND1), a member of the D-type cyclin determine the genotype and allelic frequencies of family (3), is the main cyclin that regulates the G1/S phase polymorphisms. Seventy-eight breast cancer patients and 84 progression. It does this by phosphorylating and inactivating age-matched healthy controls were included in the study. the RB protein by coupling with cyclin-dependent kinase 4 Results. Frequencies of CT genotype and T allele of CDKN1B (CDK4), which may induce the release of the transcription were found to be higher in breast cancer patients than in factor E2F from the RB protein and start the function of DNA controls (p=0.013, OR: 1.514 95% CI: 1.086-2.114.15; synthesis (4). However, cyclin D1 overexpression may result p=0.007, OR=1.496; 95% CI: 1.111-2.014, respectively). The in the phosphorylation of RB protein, in advance, frequency of AA genotype of CCND1 was decreased in uncontrolled cell growth, and the development of cancer (4). hormone receptor- (estrogen and progesterone receptors) It has been indicated that the CCND1 gene is amplified in up negative patients with breast cancer (p<0.049, OR=0.286; to 20% of breast cancer cases and overexpressed in more than 95% CI: 0.071-1.142) Conclusions. Even though CDKN1B 50% of mammary tumours (5), and this appears to be an early polymorphism appears to be an important predictive factor for event in breast cancer formation (6). In exon 4, CCND1 there breast cancer risk and CCND1 polymorphism may be a is a silent G to A substitution at nt870 (rs603965). Betticher prognostic biomarker for breast cancer, further investigations et al. (7) demonstrated that CCND1 G870A produces an with larger study groups are needed to fully elucidate the role alternative protein, transcript-b, which has been suggested to of CCND1 and CDKN1B polymorphisms in the development have a longer half-life than transcript-a (7, 8). Several studies and prognosis of breast cancer. also reported that transcript-b was a poor catalyst of RB phosphorylation or inactivation and markedly enhanced cell transformation activity compared to transcript-a (9, 10). A single nucleotide adenine-to-guanine substitution (A870G) in exon 4 of the CCND1 gene is a common Correspondence to: Emel Canbay MD, Ph.D, Atakoy 11.kisim polymorphism and is known to influence the splicing at the Gelincik Apt. B Blok Daire 12 Istanbul, 34158,Turkey. Tel: +212 6619290, Fax:+212 4880180, email: [email protected] exon 4-intron 4 boundary resulting in an alternate transcript (transcript-b) (7-9). This alternate transcript produced by the Key Words: Cyclin D polymorphism, CDKN1B polymorphism, 870A allele lacks exon 5 of CCND1 and contains a PEST- breast cancer, Turkish population rich region that is postulated to target proteins for rapid 0250-7005/2010 $2.00+.40 3093 ANTICANCER RESEARCH 30: 3093-3098 (2010) Table I. PCR and RFLP procedures and expected products of CCND1 (A870G) and CDKN1B (C79T) genes. Primers (forward and reverse) PCR PCR Restriction Restriction conditions product enzyme products CCND1 5’GTGAAG TTCATTTCCAATCCGC-3 25 μl of PCR 167 NciI A/A: 167 (23)* 5’GGGACATCACCCTCACTTAC-3’ mixture: A/G: 167, 145 16 mM of each dNTP, G/G: 145 100 pmol/μl of each primer, 15 mM of MgCl2, 1 U Taq polymerase ’ 35 cycles: 94˚C 45 s, 55˚C 45 s, 72˚C 45 s CDKN1B 5’ TGGCTCGTCGGGGTCT 3 ‘ 25 μl of PCR 195 HaeIII C/C: 129 (20) * 5’ CCATCCGCTCCAGGCT 3’ mixture: C/T: 129, 66 200 mM dNTP, T/T: 195 100 pmol/μl of each primer, 15 mM of MgCl2 1U Taq polymerase 35 cycles: 94˚C 30 sec, 56˚C 45 sec, 72˚ C 45 sec degradation, and which is hypothesized to be more stable concentrations, they have been shown to improve compared to the product of the 870G allele. It has been complexing between CDK4/6 and cyclin D, but still inhibit shown that transcript-b leads to a longer half-life of CCND1, CDK2–Cyclin-E (19). Loss of CDKN1B expression is also which may bypass the G1/S-checkpoint (7). a common event in breast cancer, and has been strongly Several molecular epidemiological studies have been associated with high tumour grade and poor prognosis (20). conducted to examine the association between CCND1 The V109G polymorphism of the p27 gene, CDKN1B, G870A polymorphism and breast cancer risk (11-17), but the examined by polymerase chain reaction analysis of tumour results remain inconsistent. specimens, was associated with shortened disease-free Progression through the cell cycle is governed by the survival in a subset of patients with infiltrating metastasis- activation of cyclin-dependent kinases (CDKs), and free breast cancer (19). However, association of the CDKN1B sequential activation of CDK–cyclin complexes leads to the C79T polymorphism and breast cancer risk has been phosphorylation and inactivation of RB, allowing analysed previously only in two studies (21, 22). transcription of cell cycle genes by the E2F family of The aim of this study was to investigate the possible transcription factors (18). This process is kept in check by correlation between the polymorphisms of CCND1 G870A inhibitors (CKIs). These are referred to by various names, and CDKN1B C79T and development of breast cancer. but are generally classified into two groups: inhibitors of kinase 4 (INK4) and CDK inhibitory protein/kinase inhibitor Patients and Methods protein (CIP/KIP). The INK4 groups includes CDKN2A (INK4A/p16 and ARF/p14), CDKN2B (INK4B/p15), Study population. The patient group consisted of 78 consecutive CDKN2C (INK4C/p18) and CDKN2D (INK4D/p19). These breast cancer patients with median age 52.5 ( range 30-79) years, bind to both CDK4 and CDK6 to prevent their association who were admitted to Istanbul University Cerrahpasa Medical Faculty, Department of General Surgery, Breast Services. The with cyclin D. The CIP/KIP group comprises CDKN1A control group consisted of age- and sex-matched healthy subjects. (WAF1/p21/CIP1) and CDKN1B (KIP1/p27), which form The control subjects were randomly selected among volunteer blood heterotrimeric complexes with the G1 to S transition CDKs. donors. Subjects with a personal or family history of any cancer and The CIP/KIP proteins do not affect cyclin binding. At low chronic diseases such as cardiovascular or cerebrovascular disease, 3094 Canbay et al: CCND1 and CDKN1B in Breast Cancer diabetes mellitus, hypertension, or renal disease were excluded from Table II. Characteristics of patients with breast cancer. the study. Control subjects were not taking any regular medication at time of the study. Eighty-four healthy women, presenting a Parameters Patients (n=78) Controls (n=84) median age 46 (range 30-81) years, were used as a control group. Informed consent was obtained from all participants. Breast cancer n%n% patients had previously undergone appropriate surgery. Questionnaires, medical records, and pathological reports were used Age (years) to confirm the diagnosis and cancer status. This study protocol was <40 22 28.2 31 36.9 approved by the Local Ethical Committee at Istanbul University ≥40 56 71.8 53 63.1 Smoking status Cerrahpasa Medical School. Yes 15 20.7 22 38.1 No 59 79.3 72 61.2 Polymorphism Analysis. This study was carried out in the Alcohol consumption Department of Molecular Medicine, Istanbul University Yes 1 1.4 2 2.4 Experimental Medicine and Research Institute. Genomic DNA No 73 98.6 82 97.6 was extracted from peripheral whole blood containing EDTA Family history according to the salting-out technique (23). Genotyping was Yes 5 6.8 performed by polymerase chain reaction (PCR) and restriction No 69 93.2 fragment length polymorphism (RFLP), the procedures of which Histopathology are given in Table I (22, 23). The appropriate primers were used Ductal 67 85.7 to amplify the corresponding gene of the subjects by PCR. The Lobular 6 7.8 reaction products were digested by using the appropriate enzyme Mixed 4 5.2 at 37˚C. The digested products were analysed on 3% agarose gel Other 1 1.3 stained with ethidium bromide and examined under Tumour stage transillumination. Each gel was read by two observers unaware T1 19 24.3 of the subject’s status. If there was any conflict, samples were T2 53 67.9 repeated. The expected results after restriction for each gene are T3 3 3.9 also given in Table I.