ANTICANCER RESEARCH 29: 1271-1274 (2009)

Association of GNB4 Intron-1 Haplotypes with Survival in Patients with UICC Stage III and IV Colorectal Carcinoma

KATHRIN RIEMANN1, HENRIKE STRUWE1, HAKAN ALAKUS2, BRIGITTE OBERMAIER3, KLAUS J. SCHMITZ4, KURT W. SCHMID4,5 and WINFRIED SIFFERT1,5

1Institute of Pharmacogenetics, University Hospital Essen; 2Department of Visceral and Vascular Surgery, University of Cologne; 3Eurofins Medigenomix GmbH, Martinsried; 4Institute of Pathology and Neuropathology, University Hospital Essen; 5Member of the West German Cancer Centre Essen (WTZE), Germany

Abstract. Background: Polymorphisms in encoding (4), as well as the regulation of cell growth subunits of heterotrimeric G have been repeatedly and proliferation via Ras activation (5). associated with the course of cancer. As previously shown, Individual genetic variations in genes encoding G- intron 1 of GNB4 harbours distinct haplotype blocks and subunits have already been associated with the course of block 1 is associated with survival and disease progression cancer, including a common single nucleotide polymorphism in urothelial bladder cancer. This study investigated whether (SNP), C825T, in the GNB3 encoding the Gβ3 subunit, haplotype block 2 is associated with survival in colorectal which has been related to three different cancer entities (6- cancer patients. Patients and Methods: The haplotype 8). In addition, two distinct haplotype blocks in intron 1 of tagging polymorphism of GNB4 haplotype block 2 was the G-protein subunit-β4 gene (GNB4, MIM610863) have genotyped in 136 colorectal cancer patients and associated recently been described. Haplotype block 1 was with demographic and clinical data and survival. Results: demonstrated to be a functional variation leading to Haplotype block 2 is associated with survival in colorectal diplotype-dependent different Gβ4 amounts and was cancer patients. Patients with advanced tumour stages associated with survival and disease progression in urothelial carrying the 2*1 haplotype revealed decreased survival (HR carcinoma (9). However, haplotype block 2, which shows no 2.04, 95% CI 0.91-3.69). In multivariate analysis, the linkage to block 1, could not be related to bladder cancer. diplotypes were independent prognostic factors. Conclusion: Therefore, the aim of this paper is to clarify whether this Intron-1 haplotypes of GNB4 might be predictive markers for GNB4 intron-1 haplotype block 2 might be correlated to a survival of patients with advanced colorectal cancer, thus different cancer entity, colorectal carcinoma. influencing the clinical management of these patients. Patients and Methods Heterotrimeric G proteins are key players in the from membrane receptors to intracellular effectors. Patients. All 136 patients with colorectal carcinoma underwent Activated -coupled receptors result in the activation surgery in the Department of General Surgery at the Klinikum of many signal transduction cascades via the α subunits and Herford between 1996 and 1998. The tissue was stored embedded in the several βγ dimers (1). Next to the well-described α- paraffin. A complete follow-up (mean 43 months) of all patients was obtained. Histopathological diagnosis was made according to mediated signalling, βγ-mediated effects have become the WHO classification and the guidelines of the UICC. In increasingly elucidated. These events include the regulation of compliance with the recommendations of the German Surgical ion channels (2), certain isoforms of (3) or Oncology Working Group (CAO), patients with UICC stage II colorectal carcinoma did not receive adjuvant treatment, while patients with stage-III and -IV tumours received a standardised chemotherapy consistent with the Mayo Clinic protocol. The study Correspondence to: Dr. rer. nat. Kathrin Riemann, Institute of was performed according to the declaration of Helsinki and Pharmacogenetics, University Hospital Essen, Hufelandstr. 55, approved by the Ethics Committee of the University Hospital Essen. 45147 Essen, Germany. Tel: +49 2017233459, Fax: +49 2017235968, e-mail: [email protected] Genotyping. Genomic DNA from paraffin-embedded tissue of the patients was extracted using standard methods (QIAamp; Qiagen, Key Words: G protein, GNB4, haplotype, SNP, colorectal cancer. Hilden, Germany). Haplotype block 2 consists of four SNPs

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Table I. Demographic and clinical characteristics at primary diagnosis Table II. Multivariate Cox regression models for survival in patients and diplotype distribution of GNB4 block 2 in patients with colorectal with colorectal cancer. cancer. Tumour-related death All 2*1/2*1 2*1/2*2 2*2/2*2 p All cases Only UICC III+IV n (% ) 136 42 (30.9) 66 (48.5) 28 (20.6) Hazard ratio p Hazard ratio p Gender (M/F) 71/65 22/20 35/31 14/14 0.314 (95% CI) (95% CI) Mean age at 67.90 68.76 66.26 70.50 0.171 diagnosis Gender (years) (±10.6) (±10.1) (±11.1) (±9.7) Male 1# 1# ≤65 49 (36.0) 14 (28.6) 27 (55.1) 8 (16.3) Female 1.30 (0.76-2.23) 0.341 1.09 (0.57-2.09) 0.788 >65 87 (64.0) 28 (32.2) 39 (44.8) 20 (23.0) 0.427 Age at diagnosis Tumour site ≤65 years 1# 1# Colon 72 (55.8) 27 (37.5) 32 (44.4) 13 (18.1) >65 years 0.76 (0.44-1.33) 0.335 1.05 (0.55-2.01) 0.875 Rectum 57 (44.2) 14 (24.6) 31 (54.4) 12 (21.1) 0.545 Tumour Grade localisation 1 57 (43.8) 9 (15.8) 33 (57.9) 15 (26.3) Rectum 1# 1# 2 59 (45.4) 24 (40.7) 26 (44.1) 9 (15.3) Colon 1.48 (0.87-2.53) 0.153 2.17 (1.05-4.49) 0.037 3 14 (10.8) 6 (42.9) 4 (28.6) 4 (28.6) 0.024 Stage Tumour UICC I+II 1# invasion UICC III+IV 2.67 (1.52-4.68) 0.001 pT1 11 (8.2) 3 (27.3) 7 (63.6) 1 (9.1) UICC III 1# pT2 16 (11.9) 5 (31.3) 7 (43.8) 4 (25.0) UICC IV 3.08 (1.62-5.89) 0.001 pT3 75 (56.0) 19 (25.3) 39 (52.0) 17 (22.7) Block 2 pT4 32 (23.9) 14 (43.8) 12 (37.5) 6 (18.8) 0.524 2*2/2*2 1# 1# Lymph 2*1 1.31 (0.68-2.35) 0.427 1.90 (0.77-4.73) 0.046 node status pN0 66 (49.6) 18 (27.3) 31 (47.0) 17 (25.8) #, Reference group. pN+ 67 (50.4) 23 (34.3) 34 (50.7) 10 (14.9) 0.279 Metastases pM0 106 (78.5) 29 (27.4) 53 (50.0) 24 (22.6) SPSS 13.0 (SPSS, Chicago, IL, USA) or Graphpad Prism 4.0 pM1 29 (21.5) 12 (41.4) 13 (44.8) 4 (13.8) 0.293 (Graphpad Software, San Diego, CA, USA). Differences were UICC stage p I 19 (14.2) 6 (31.6) 10 (52.6) 3 (15.8) considered to be statistically significant at <0.05. II 41 (30.6) 11 (26.8) 18 (43.9) 12 (29.3) III 45 (33.6) 12 (26.7) 24 (53.3) 9 (20.0) Results IV 29 (21.6) 12 (41.4) 13 (44.8) 4 (13.8) 0.623 GNB4 block-2 haplotype frequencies were 55.1% for 2*1 Data are numbers with percentage given in brackets or means (±SD). Categorical variables were analysed by χ2 statistics, continuous and 44.9% for 2*2 in patients with colorectal carcinoma. parameters by analysis of variance. M, male; F, female. Correlations between diplotypes and demographic as well as clinico-pathological data are presented in Table I. During the follow-up period 68 patients (50% ) died. Diplotype- (rs6443650, rs7618348, rs6443649, rs6443648) in complete linkage dependent survival of patients was not different (p=0.213; disequilibrium (D’=1, r2=1) within a 337-bp region in intron 1 of Figure 1a). Interestingly, an association (p=0.029; Figure 1b) GNB4 (9). Genotypes for the haplotype tagging SNP rs6443650 existed in the subgroup of patients with advanced tumour were determined by restriction fragment analysis. A 142-bp product stages (UICC III/IV) with an apparent gene-dose effect. was amplified by polymerase chain reaction (PCR) using primers Patients harbouring the 2*1 haplotype were at higher risk for GNB4_2mut_f (5’-agagtgatacatgaggccag-3’) and GNB4_2_r (5’- aattagagcaactagagatcca-3’) which introduce a restriction site for tumour-related death than individuals with 2*2/2*2 (HR BslI. The GG genotype displayed two fragments of 119 and 23 bp 2.04, 95% CI 0.91-3.69). After five years, only 14.3% and after digestion representing diplotype 2*1/2*1 (GGCT/GGCT), the 32.4% of these patients with at least one 2*1 allele were still TT genotype showed the uncut 142-bp fragment representing alive compared to 54.5% of 2*2/2*2 carriers. diplotype 2*2/2*2 (TATA/TATA), and the heterozygous genotype While multivariate analysis for all patients showed that the revealed all three fragments representing both haplotypes 2*1/2*2 UICC stage was the only independent predictive factor for (GGCT/TATA). survival, in the subgroup of patients with advanced stages, Statistical analysis. Survival was plotted by the Kaplan-Meier three prognostic factors turned out to be independent. Here, method and evaluated by the linear log rank test. The impact of the haplotype combination also revealed statistical prognostic factors was calculated by backward stepwise multivariate significance as an independent risk factor (2*1 vs. 2*2/2*2: Cox regression models. Statistical analyses were performed using HR 1.90, 95% CI 0.77-4.73, p=0.046, Table II).

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impacts mainly on advanced cancer. Since this subgroup (UICC III/IV) represents patients who received standard chemotherapy, the haplotype block 2 might be related to response to therapy. Verifying this hypothesis would require a test group consisting of historical samples with advanced UICC stages but without chemotherapeutic treatment for comparison of survival effects. However, the question of functional consequences of the block-2 haplotypes arises. As previously described, unlike block 1, reporter activity of block-2 haplotypes did not differ (9). Since the promoter of GNB4 did not contain any SNPs that might influence gene expression, haplotype block 2 could be linked to functional variations outside the gene . GNB4 is located in reverse direction on 3q26.32- 33 adjacent to genes encoding mitochondrial proteins (e.g. MFN1, NDUFB5) which contribute to mitochondrial integrity. Mitochondrial dynamics have been implicated in the regulation of . Induction of cell death is sometimes associated with the fragmentation of the mitochondrial network (10). Therefore, expression of these genes might be important for mitochondrial function and consequently for the well-being of the cell. A preliminary search in the HapMap data revealed several SNP candidates in linkage disequilibrium that should be targeted in further studies to elucidate the suggested relationship to the haplotype block 2. In summary, the second association of GNB4 intron-1 haplotypes with the prognosis of a tumour disease is hereby presented. These results, of course, have to be validated in Figure 1. Kaplan-Meier plots for the investigated patient sample. (a) independent studies including investigations about linked Survival of all patients with colorectal cancer and (b) survival of patients functional SNPs which might be causally responsible for this with UICC stage III and IV colorectal cancer over a follow-up period of association and also target therapy response. 92 months dependent on different diplotypes of GNB4 block 2. Acknowledgements

This study was supported by the German Federal Ministry of Discussion Education and Research (BioChancePLUS).

In this study, whether the haplotype block 2 located in intron References 1 of GNB4 is associated with survival in patients with colorectal cancer was examined. No relationship between 1 Cabrera-Vera TM, Vanhauwe J, Thomas TO, Medkova M, survival and haplotype combinations could be shown when Preininger A, Mazzoni MR and Hamm HE: Insights into G all patients were analysed regardless of tumour invasiveness. protein structure, function, and regulation. Endocr Rev 24: 765- 781, 2003. Interestingly, a strong association existed in the subgroup of 2 Ofer WG: G protein regulation of channels. In: Handbook of patients with UICC III and IV colorectal tumours. Almost Cell Signaling. Bradshaw RA, Dennis EA (eds.). Boston, four times as many patients with the homozygous 2*2 Academic Press, pp. 667-670, 2004. haplotype were still alive after a 5-year follow-up compared 3 Exton JH: Regulation of phosphoinositide phospholipases by to patients carrying 2*1/2*1. This correlation is similar to hormones, neurotransmitters, and other agonists linked to G observations made in urothelial carcinoma, which showed an proteins. Annu Rev Pharmacol Toxicol 36: 481-509, 1996. association of the block-1 haplotypes with survival and 4 Sunahara RK, Dessauer CW and Gilman AG: Complexity and diversity of mammalian adenylyl cyclases. Annu Rev Pharmacol disease progression in the subset of muscle-invasive tumours Toxicol 36: 461-480, 1996. (9). The conformance of diplotype-dependent differences in 5 Schwindinger WF and Robishaw JD: Heterotrimeric G-protein disease course of diverse tumours with advanced stages betagamma-dimers in growth and differentiation. Oncogene 20: points to a common characteristic feature of GNB4, which 1653-1660, 2001.

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6 Sheu SY, Gorges R, Ensinger C, Ofner D, Farid NR, Siffert W 9 Riemann K, Struwe H, Eisenhardt A, Obermaier B, Schmid KW and Schmid KW: Different genotype distribution of the GNB3 and Siffert W: Characterization of intron-1 haplotypes of the G C825T polymorphism of the G protein beta3 subunit in adenoma protein β4 subunit gene – association with survival and and differentiated thyroid carcinomas of follicular cell origin. J progression in patients with urothelial bladder carcinoma. Pathol 207: 430-435, 2005. Pharmacogenet Genom 18: 999-1008, 2008. 7 Nückel H, Frey U, Aralh N, Dürig J, Dührsen U and Siffert W: 10 Chen H, Dettmer SH, Ewald AJ, Griffin EE, Fraser SE and Chan The CC genotype of the C825T polymorphism of the G protein DC: Mitofusins Mfn1 and Mfn2 regulate mitochondrial fusion beta3 gene (GNB3) is associated with a high relapse rate in and are essential for embryonic development. J Cell Biol 160: patients with chronic lymphocytic leukemia. Leuk Lymphoma 189-200, 2003. 44: 1739-1743, 2003. 8 Eisenhardt A, Siffert W, Rosskopf D, Musch M, Roggenbuck U, Jöckel KH, Rübben H and Lümmen G: Association study of the G-protein beta3 subunit C825T polymorphism with disease Received December 10, 2008 progression in patients with bladder cancer. World J Urol 23: Revised January 15, 2009 279-286, 2005. Accepted February 2, 2009

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