Andreas Eisenhardt

The T393C polymorphism of the Gαs gene (GNAS1) is a novel prognostic marker in bladder cancer

The G protein Gαs pathway is linked to proapoptotic signaling in cancer cell lines. To assess the role of the GNAS1 locus encoding Gαs as a genetic factor for disease progression of transitional cell carcinoma (TCC) of the bladder, we genotyped the synonymous T393C polymorphism in 254 patients with TCC (minor allele frequency: 0.43) to examine a potential association between genotypes and disease progression. Using Kaplan-Meier estimates to calculate 5-year probabilities of follow-up, we could show that progression-free survival, metastasis-free survival, and cancer-specific survival was significantly increased in TT genotypes (56%, 84%, 82%) compared with CC genotypes (35%, 53%, 58%). In multivariate Cox proportional hazard analysis, the T393C polymorphism was an independent prognostic factor for clinical outcome. Homozygous CC patients were at highest risk for progression [odds ratio (OR), 1.94; P = 0.020], metastasis (OR, 3.49; P = 0.005), and tumor-related death (OR, 2.49; P = 0.031) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. Real-time PCR analysis of urothelial tumor tissue as well as adipose and heart tissue revealed that Gαs mRNA expression was highest in TT genotypes, indicating a proapoptotic effect in these genotypes. In conclusion, the GNAS1 T393C status associated with differential Gαs mRNA expression is a novel independent prognostic marker for clinical outcome supporting a functional role of Gαs in bladder cancer progression.

The GNAS1 T393C Polymorphism Predicts Survival in Patients with Clear Cell Renal Cell Carcinoma

Purpose: G proteins mediate signaling from cell surface receptors to specific intracellular proteins. In vitro cancer cell line studies revealed a link between the Gαs protein and proapoptotic processes. We have recently shown that TT genotypes of the GNAS1 T393C polymorphism display increased transcription of Gαs and a more favorable clinical course in bladder and colorectal cancer patients compared both with TC or CC genotypes. Experimental Design: In the present study, 150 patients with clear cell renal cell carcinoma surgically treated by nephrectomy with curative intent were retrospectively genotyped to elucidate a potential association between T393C genotypes and clinical outcome. Results: The C-allele frequency in the renal cell carcinoma patient group was 0.51, which is not significantly different from that of a healthy blood donor group. Kaplan-Meier curves for tumor progression, development of metastasis, and tumor-related death showed a significant association of the T393C polymorphism with outcome (5-year cancer-specific survival rates: TT, 91%; TC, 81%; CC, 69%; P = 0.015). Multivariate Cox proportional analysis of a 10-year follow-up confirmed the T393C polymorphism as an independent prognostic factor in clear cell renal cell carcinoma. Homozygous CC patients were at highest risk for progression (hazard ratio, 2.48; P = 0.009) or tumor-related death (hazard ratio, 3.15; P = 0.018) compared with T-allele carriers. Conclusion: Our results show that besides tumor stage, lymph node status, and tumor grade, the GNAS1 T393C status is a novel independent host factor for disease progression in patients with clear cell renal cell carcinoma and provides further evidence for the T393C polymorphism as a general prognostic tumor marker.

Identification and characterization of G beta 3s2, a novel splice variant of the G-protein beta 3 subunit.

The T-allele of a polymorphism (C825T) in the gene for the G-protein beta 3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of G beta 3s, a splice variant of GNB3. G beta 3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional G beta 3 splice variant referred to as G beta 3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. G beta 3s2 is generated by alternative splicing using non-canonical splice sites. G beta subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for G beta 3s2 are lacking 129 bp of the coding sequence of the wild-type G beta 3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of G beta 3s. Co-immunoprecipitation analyses indicated that G beta 3s2 dimerizes with different G gamma subunits, e.g. G gamma 5, G gamma 8(C) and G gamma 12. In Sf9 insect cells, expression of G beta 3s2 together with G gamma 12 enhances receptor-stimulated activation of G alpha(i2). Expression of G beta 3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that G beta 3s2 is a biologically active G beta variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.

Regulatory BCL2 promoter polymorphism (-938C>A) is associated with adverse outcome in patients with prostate carcinoma.

Molecular markers predictive of prostate cancer prognosis are urgently needed. Overexpression of the antiapoptotic protein, Bcl‐2, has repeatedly been shown to be associated with adverse outcome in this malignancy. We hypothesized that a regulatory BCL2 −938C>A promoter polymorphism, which significantly affects promoter activity and Bcl‐2 expression in different malignancies, may influence survival. Reporter assays and electrophoretic mobility shift assays reveled that the −938C>A BCL2 promoter polymorphism significantly affects promoter activity and transcription factor binding in prostate cancer cells. Significantly higher BCL2 mRNA expression was observed in primary prostate carcinomas derived from patients with the AA, compared to CC, genotype. Survival analysis showed that the −938AA genotype was an independent, unfavorable prognostic factor for relapse‐free survival in a primary cohort of 142 patients and in an independent replication cohort of 148 patients, with hazard ratios (HR) of 4.4 (95% CI, 1.3–15.1; p = 0.018) and 4.6 (95% CI, 1.5–14.2; p = 0.009). Furthermore, the −938AA genotype was independently associated with worse overall survival in the replication series, with a HR of 10.9 (95% CI, 1.2–99.3; p = 0.034). We conclude that the BCL2 −938C>A polymorphism is an independent predictor of relapse‐free and overall survival in patients with prostate cancer. The BCL2 −938C>A polymorphism should be evaluated prospectively and may also have promise in assisting optimal patient choice for treatment with BCL2‐targeted drugs already in evaluation for prostate cancer treatment.

Association study of the G-protein β3 subunit C825T polymorphism with disease progression in patients with bladder cancer

The T-allele in the GNB3 C825T polymorphism has been associated with increased cell migration, a prerequisite for metastasis. In this study we investigated a potential association of the C825T-allele status and disease progression in patients with transitional cell carcinoma of the bladder (TCC). Genotyping of the GNB3 C825T polymorphism was performed in 389 patients with transitional cell carcinoma of the bladder and in 104 control subjects and clinical follow-up was worked up in 339 patients. Genotype distribution in 389 patients with bladder cancer was comparable to genotype distribution of the control group. There was no association of GNB3 C825T genotype with tumor stage or grade, but follow-up analysis in the subgroup of non-smokers revealed a shorter time to metastasis in 825T-allele carriers compared to individuals homozygous CC. The genotype of the GNB3 C825T polymorphism appears to influence the biological behavior of tumor disease in non-smoking TCC patients.

Genetic Risk Factors for Erectile Dysfunction and Genetic Determinants of Drug Response - on the Way to Improve Drug Safety?

Abstract.Association studies give hint for the fact that the risk to develop cardiovascular disorders such as hypertension or coronary heart disease is influenced by the genotype in single nucleotide polymorphisms (SNPs). Considering the close relationship in the pathophysiology of these diseases and erectile dysfunction (ED), the analysis of the association of genotypes in SNPs and ED stands to reason. In an analysis of ED patients and their genotypes in the GNB3 C825T, the ACE I/D and the NOS3 G894T polymorphisms, there was no evidence for influence of the genotypes on the susceptibility to develop ED. At the same time, a significant variation in drug response to sildenafil dependent on the genotypes in the GNB3 C825T and ACE I/D polymorphisms was demonstrated. In the group of GNB3 825C allele carriers, only 50% of patients showed a positive response, while > 90% of the patients genotype TT responded adequately. In parallel, only 50% of ACE D allele carriers showed a positive response to sildenafil in contrast to men genotype II in the ACE I/D polymorphism, who had a response rate of 75%. Considering cardiovascular side effects under sildenafil treatment, it would be interesting to determine if genetic factors have an impact on the side effect profile of this drug.Zusammenfassung.Die erektile Dysfunktion ist eine häufige Erkrankung des alternden Mannes, die in milder Ausprägung bis zu 52% der Männer zwischen 40 und 70 Jahren betrifft. Etablierte Risikofaktoren für die Entwicklung einer erektilen Dysfunktion sind fortschreitendes Alter, arterielle Hypertonie, Arteriosklerose, Diabetes mellitus, Hypercholesterinämie und Nikotinabusus. Die koronare Herzerkrankung und die periphere arterielle Verschlusskrankheit weisen ein ähnliches Risikofaktorenprofil auf, und es gibt Hinweise auf verwandte Elemente in der Pathophysiologie dieser Erkankungen, wie z.B. die endotheliale Dysfunktion. Untersuchungen lassen darauf schließen, dass das individuelle kardiovaskuläre Risiko durch die Genotypen in Polymorphismen determiniert wird, bei denen eine Assoziation z.B. mit der Hypertonie oder der koronaren Herzerkrankung nachgewiesen werden konnte. In einer Gruppe von 113 Patienten mit erektiler Dysfunktion wurden das Genotypenprofil der GNB3-C825T-, ACE-I/D- und NOS3-G894T-Polymorphismen und der Zusammenhang mit dem Ansprechen auf Sildenafil untersucht. Hierbei zeigte sich kein Einfluss der Genotypen in diesen drei Polymorphismen auf das Risiko zur Prädisposition einer erektilen Dysfunktion. Interessanterweise wurde das Ansprechen auf Sildenafil durch die Genotypen im GNB3-C825T- und ACE-I/D-Polymorphismus deutlich beeinflusst. Die Patienten Genotyp TT im GNB3-C825T-Polymorphismus sprachen zu 90,9% auf Sildenafil an, während nur ca. 50% der Patienten Genotyp TC oder CC ein positives Ansprechen aufwiesen. Parallel waren unter den Patienten Genotyp II im ACE-I/D-Polymorphismus 75% Sildenafil-Responder zu beobachten, unter den Patienten Genotyp ID und DD dagegen nur jeweils ca. 50%. Obwohl Sildenafil ein gutes Sicherheitsprofil aufweist, wurde insbesondere bei Risikopatienten das Auftreten von kardiovaskulären Nebenwirkungen unter der Therapie mit diesem oralen Phosphodiesterasehemmer beschrieben. Weitere Untersuchungen werden Aufschluss darüber geben, ob das Auftreten kardiovaskulärer Ereignisse durch genetische Faktoren determiniert wird.

Does cigarette smoking influence the survival of patients with prostate cancer

ZusammenfassungDas Prostatakarzinom hat sich in den vergangenen Jahren weltweit zu einer der häufigsten Todesursachen entwickelt. Daher ist es wichtig, mögliche Risikofaktoren für diese maligne Tumorerkrankung zu ermitteln. Neben den Risikofaktoren, die zu einem erhöhten Erkrankungsrisiko führen, sollte das Augenmerk auch auf Faktoren gerichtet werden, die den Erkrankungsverlauf beeinflussen können. In unserer Untersuchung konnten wir einen signifikant schlechteren Erkrankungsverlauf für das Prostatakarzinom bei Patienten nachweisen, die zum Zeitpunkt der Erstdiagnose ihrer Tumorerkrankung Zigaretten rauchten. Trotz gleicher Ausgangsbedingungen bezüglich Staging, Grading und Ausgangs-PSA-Wert, haben die Raucher der untersuchten Gruppe ein 3fach erhöhtes Risiko, tumorbedingt zu versterben. Dieser Effekt ist möglicherweise auf durch das Rauchen ausgelöste Stoffwechselveränderungen zurückzuführen, die sowohl das Tumorwachstum als auch die Metastasenbildung begünstigen.AbstractIn the last few years, prostate cancer has become one of the most common causes of mortality worldwide. It is therefore important to detect possible risk factors for this malignant disease. Besides risk factors which increase incidence, attention should be paid to factors which have a possible influence on the course of the disease. In our analysis, we demonstrate a worse course for the disease in patients with prostate cancer who smoked cigarettes at the time of first diagnosis. In spite of comparable staging, grading and PSA values at the time of primary diagnosis, individuals who smoked had a threefold higher risk of dying from prostate cancer. This effect is probably caused by metabolic changes which are activated by cigarette smoking and promote tumor growth and the development of metastases.

Characterization of intron-1 haplotypes of the G protein β4 subunit gene : association with survival and progression in patients with urothelial bladder carcinoma

Purpose Polymorphisms in genes encoding subunits of heterotrimeric G proteins have been repeatedly associated with various cancers. As G&bgr;&ggr; signaling is presumed to be involved in proliferation and invasion processes, we analyzed genetic variations in regulatory regions of GNB4, which encodes the G&bgr;4 subunit, for their potential influence on cancer progression. Experimental Design We characterized the promoter of GNB4 and screened the promoter as well as exon 1 and intron 1 for single nucleotide polymorphisms by sequencing 100 healthy controls. Following a haplotype analysis, we determined the functional impact upon gene expression of the defined haplotypes by reporter assays, electrophoretic mobility shift assay and western blot. In addition, these haplotypes were tested for their relation to the disease course of urothelial bladder cancer. Results Whereas the promoter of GNB4 revealed no polymorphisms, 33 single nucleotide polymorphisms located in exon 1 and intron 1 were identified and together with a common exon-4 polymorphism implemented in haplotype analysis, which resulted in the determination of distinct haplotype blocks. Reporter activity was haplotype-dependently different (P=0.001). 1*1/1*1 showed increased G&bgr;4 protein (P=0.003), and bladder cancer patients carrying this diplotype displayed more progressive disease (P=0.046) and a significantly increased mortality (P=0.046). In multivariate analysis,the diplotypes were independent prognostic factors for survival and progression. Conclusion Intron-1 haplotypes of GNB4 may, thus, serve as predictive markers for progression and survival of patients suffering from bladder cancer.

Characterization of the splice variant Gβ3v of the human G-protein Gβ3 subunit

Abstract A polymorphism (C825T) in the gene of the G-protein Gβ3 (GNB3) has been the subject of numerous studies which have shown that the 825T-allele is associated with several cardiovascular and metabolic disorders. Furthermore, the T-allele is associated with the occurrence of the splice variant Gβ3s which has been implicated in the pathogenesis of these disorders. Here, we characterise a novel splice variant of GNB3, termed Gβ3v, which is generated by alternative splicing of parts from intron 9 as a novel exon 10a. Gβ proteins belong to the superfamily of propeller proteins composed of seven regular WD-domains. In Gβ3v, four of these WD-domains are retained but the protein has a novel C terminus. Gβ3v forms dimers with Gγ3 and Gγ12 but these Gβγ complexes do not stimulate phospholipase C-β2. Thus, a physiological role of Gβ3v remains to be established. Gβ3v transcripts are detectable in a wide variety of cells and tissues including fibroblasts, B lymphoblasts, retinoblastoma cells, retina, brain, umbilical cord and colon. However, there is no association with an allele of the GNB3 C825T polymorphism, which suggests that Gβ3v does not contribute to the complex phenotype observed in association with the 825T-allele.

Beeinflusst Rauchen das Überleben von Prostatakarzinompatienten

ZusammenfassungDas Prostatakarzinom hat sich in den vergangenen Jahren weltweit zu einer der häufigsten Todesursachen entwickelt. Daher ist es wichtig, mögliche Risikofaktoren für diese maligne Tumorerkrankung zu ermitteln. Neben den Risikofaktoren, die zu einem erhöhten Erkrankungsrisiko führen, sollte das Augenmerk auch auf Faktoren gerichtet werden, die den Erkrankungsverlauf beeinflussen können. In unserer Untersuchung konnten wir einen signifikant schlechteren Erkrankungsverlauf für das Prostatakarzinom bei Patienten nachweisen, die zum Zeitpunkt der Erstdiagnose ihrer Tumorerkrankung Zigaretten rauchten. Trotz gleicher Ausgangsbedingungen bezüglich Staging, Grading und Ausgangs-PSA-Wert, haben die Raucher der untersuchten Gruppe ein 3fach erhöhtes Risiko, tumorbedingt zu versterben. Dieser Effekt ist möglicherweise auf durch das Rauchen ausgelöste Stoffwechselveränderungen zurückzuführen, die sowohl das Tumorwachstum als auch die Metastasenbildung begünstigen.AbstractIn the last few years, prostate cancer has become one of the most common causes of mortality worldwide. It is therefore important to detect possible risk factors for this malignant disease. Besides risk factors which increase incidence, attention should be paid to factors which have a possible influence on the course of the disease. In our analysis, we demonstrate a worse course for the disease in patients with prostate cancer who smoked cigarettes at the time of first diagnosis. In spite of comparable staging, grading and PSA values at the time of primary diagnosis, individuals who smoked had a threefold higher risk of dying from prostate cancer. This effect is probably caused by metabolic changes which are activated by cigarette smoking and promote tumor growth and the development of metastases.