Peter Würl

Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma

The RASSF1A tumor suppressor is involved in regulation of apoptosis and cell cycle progression. RASSF1A is localized to microtubules and binds the apoptotic kinases MST1 and MST2. It has been shown that this interaction is mediated by the Sav‐RASSF‐Hpo domain, which is an interaction domain characterized for the Drosophila proteins Sav (human WW45), Hpo (human MST1 and MST2) and Warts/LATS (large tumor suppressor). Previously, we have reported that RASSF1A hypermethylation occurs frequently in soft tissue sarcoma and is associated with an unfavorable prognosis for cancer patients. In our study, we performed methylation analysis of the CpG island promoter of MST1, MST2, WW45, LATS1 and LATS2 in soft tissue sarcomas by methylation‐specific PCR. No or a very low methylation frequency was detected for WW45, LATS1 and LATS2 (<7%). In 19 out of 52 (37%) sarcomas, a methylated promoter of MST1 was detected and 12 out of 60 (20%) samples showed methylation of the MST2 promoter. Methylation status of MST1 was confirmed by bisulfite sequencing. In tumors harboring a methylated promoter of MST1, a reduction of MST1 expression was observed by RT‐PCR. In leiomyosarcomas, MST1 and MST2 or RASSF1A methylation were mutually exclusive (P = 0.007 and P = 0.025, respectively). Surprisingly, a significantly increased risk for tumor‐related death was found for patients with an unmethylated MST1 promoter (P = 0.036). In summary, our results suggest that alteration of the Sav‐RASSF1‐Hpo tumor suppressor pathway may occur through hypermethylation of the CpG island promoter of MST1, MST2 and/or RASSF1A in human sarcomas.

Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53.

Survivin, a member of the inhibitors-of-apoptosis gene family, is overexpressed in many tumor types. Survivin is a prognostic marker of soft-tissue sarcomas, but the downregulation of survivin expression and the possible dependency of survivin downregulation on p53 in these tumors have not been investigated. Therefore, we applied small interfering RNA (siRNA) to knock down the expression of survivin in five human sarcoma cell lines with wild-type or mutant p53 alleles. Compared with survivin mRNA expression in the nonsense siRNA-treated sarcoma cell lines, expression after treatment with survivin-specific siRNA was reduced by 73–88%; survivin protein expression was reduced by 52–81%. This finding was coupled with a reduction in clonogenic survival ranging from 65–86%. However, less than 10% of cells treated with survivin-specific siRNA underwent apoptosis. Cell-cycle and morphologic analyses showed that after a dramatic increase in the number of treated cells in the G2/M phase, some of the cells became polyploid; this result indicates that mitosis of a substantial number of treated cells was incomplete. Our findings suggest that survivin-specific siRNA could be a selective treatment to kill sarcoma cells regardless of the presence or absence of wild-type p53 alleles.

Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma.

Self-renewal is considered as a common property of stem cells. Dysregulation of stem cell self-renewal is likely a requirement for the development of cancer. Hiwi, the human Piwi gene, encodes a protein responsible for stem cell self-renewal. In this study, we investigated the expression of Hiwi at the RNA level by real-time quantitative PCR in 65 primary soft-tissue sarcomas (STS) and ascertained its impact on prognosis for STS patients. In a multivariate Coxs proportional hazards regression model, we found that an increased expression of Hiwi mRNA is a significant negative prognostic factor for patients with STS (P=0.017; relative risk 4.6, 95% confidence interval (CI) 1.3–16.1) compared to medium expression of Hiwi transcript. However, a low expression of Hiwi transcript is correlated with a 2.4-fold (CI 0.7–8.0) increased risk, but this effect was not significant (P=0.17). Altogether, high-level expression of Hiwi mRNA identifies STS patients at high risk of tumour-related death. This is the first report showing a correlation between expression of a gene involved in stem cell self-renewal and prognosis of cancer patients.

High prognostic significance of Mdm2/p53 co-overexpression in soft tissue sarcomas of the extremities.

Soft tissue sarcomas are a heterogenous group of neoplasms with various histological subtypes. Up to now, no individual causal molecular markers for prognosis and therapeutic success have been identified. A tumorigenic connection between the oncogene product Mdm2 and tumor suppressor p53 is generally accepted, but their possible clinical relevance has not yet been investigated sufficiently in soft tissue sarcoma. In 86 primary soft tissue sarcoma of the extremities (RO-resected, T1/2 N0 M0), Mdm2 and p53 overexpression were investigated by immunohistochemistry. The results were adjusted to clinico-pathological characteristics and evaluated for their prognostic relevance by multivariate analysis. In Coxs multivariate analysis with stratification of Mdm2 to p53 results, we determined four groups which had different prognostic values for relapse-free and overall survival (Mdm2−/p53−<Mdm2−/p53+ <Mdm2+/p53−<Mdm2+/p53+). The most striking finding was a relative risk (rr) for overall survival of 18.77 (P=0.006) for patients with Mdm2/p53 co-overexpression (n=40). It is noticeably higher than the additive risk from both factors. Coincident Mdm2/p53 overexpression is an independent molecular marker with the highest prognostic relevance described for soft tissue sarcoma. Thus, a high risk sarcoma group has been defined which we believe requires alternative therapeutic approaches.

Co-expression of survivin and TERT and risk of tumour-related death in patients with soft-tissue sarcoma

Increased expression of survivin has been shown to be a negative predictor of survival in patients with soft-tissue sarcoma. We investigated 89 adults with soft-tissue sarcomas to ascertain the relation between co-expression of survivin and human telomerase reverse transcriptase (TERT) transcripts and prognosis. We quantified mRNA expression of survivin and TERT transcripts. Coxs proportional-hazards regression model showed co-expression of both genes to be a significant negative prognostic factor for patients with stage I to stage IV tumours (p=0 small middle dot0004; relative risk 20 small middle dot1, 95% CI 3 small middle dot8-106 small middle dot4) and for those at stage II and III (p=0 small middle dot0002; 42 small middle dot1, 6 small middle dot0-294 small middle dot9) compared with low expression of both genes. Co-expression of survivin and TERT transcripts identifies patients at high risk of tumour-related death.

Increased survivin transcript levels: An independent negative predictor of survival in soft tissue sarcoma patients

Survivin, a recently identified inhibitor of apoptosis protein (IAP), is expressed in diverse embryonic tissues and in various human cancers. We have investigated the quantitative expression of survivin mRNA by a sensitive TaqMan™‐based RT‐PCR assay in tissue samples from 94 patients with soft tissue sarcomas (STS). Survivin transcript levels were measured and normalized to GAPDH transcripts. By using a multivariate Cox regression analysis, we found an inverse correlation between the level of survivin mRNA (ratio >2 zmol survivin/amol GAPDH) and the rate of overall survival (p = 0.009, RR = 2.7). Survivin transcript variants as detected by qualitative RT‐PCR analysis were revealed in 36 of 56 STS patients (64%). Only survivin ΔEx3 and/or full‐length survivin variants but not survivin 2B were identified. Our results suggest that a higher level of survivin mRNA is an independent predictor of survival for STS patients.

The Role of the Casein Kinase 1 (CK1) Family in Different Signaling Pathways Linked to Cancer Development

The members of the casein kinase 1 (CK1) family are highly conserved and are expressed in many eukaryotes ranging from yeast to humans. Mammalian CK1 isoforms (a, ß, ?, d, e) and their splice variants are involved in diverse cellular processes including membrane trafficking, circadian rhythm, cell cycle progression, chromosome segregation, apoptosis and cellular differentiation. Mutations and deregulation of CK1 expression and activity has been linked to various diseases including neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, sleeping disorders and proliferative diseases such as cancer. In this review, we summarize the functions of CK1 and outline the participation of CK1 in signal transduction pathways linked to cancer development.

Significance of HDMX-S (or MDM4) mRNA splice variant overexpression and HDMX gene amplification on primary soft tissue sarcoma prognosis.

The product of the HDMX (or MDM4) gene is structurally related to the MDM2 oncoprotein and is also capable of interacting with the tumor suppressor protein p53. The aim of our study was to determine the amplification status of the HDMX gene and the expression of the HDMX mRNA (particularly that of the HDMX‐S splice variant) in soft‐tissue sarcomas (STS). Patients with STS were evaluated for the status of HDMX gene amplification (n = 66) and HDMX‐S mRNA expression (n = 57) within their tumors. DNA, total RNA and protein were isolated from frozen tumor tissue. We determined that the HDMX‐S splice variant transcript was predominant in a subset (14%) of tumor samples and that its expression was correlated with decreased patient survival (15 vs. 53 months, p < 0.0001, log‐rank test) and with a 17‐fold increased risk of a tumor‐related death (p < 0.0001, multivariate Coxs regression model). The tumors from these patients also expressed elevated levels of HDMX‐S protein. The HDMX gene was amplified in 17% of STSs, and the gene amplification was associated with poor prognosis (RR = 6.5, p < 0.0001). There was no correlation between the HDMX gene amplification and overexpression of the HDMX‐S splice variant. In summary, our data indicate that both the overexpression of the HDMX‐S transcript as well as HDMX gene amplification are important prognostic markers for STS.

Genomic imbalances of 7p and 17q in malignant peripheral nerve sheath tumors are clinically relevant.

We investigated 31 malignant peripheral nerve sheath tumors (MPNSTs) from 23 patients by means of comparative genomic hybridization (CGH) in order to study quantitative genomic aberrations of these tumors. Twenty‐one of the 23 patients revealed changes, with a mean value of 11 aberrations per sample (range 2–29). The minimal common regions of the most frequent gains were 8q23‐q24.1 (12 cases), 5p14 (11 cases), and 6p22‐pter, 7p15‐p21, 7q32‐q35, 8q21.1‐q22, 8q24.2‐qter, and 17q22‐qter (10 cases each). Seventeen high‐level amplifications were detected in eight of the 21 samples. In three cases, the high‐level amplifications involved 8q24.1‐qter, and in two cases each the high‐level amplifications involved regions 5p14, 7p14‐pter, 8q21.1‐q23, and 13q32‐q33. The minimal common region of frequent losses was 14q24.3‐qter (five cases). The gain of 8q as a single common change in the primary tumor, the recurrence, and the metastasis from the same patient suggests that this aberration is an early change in the tumorigenesis of MPNSTs. Comparable aberrations were observed in separate tumors of the same patients affected by Recklinghausens disease, indicating a limited number of accidental secondary changes. In sporadic MPNSTs, the most frequent gains were narrowed down predominantly to 5p, 6, 8q, and 20q, whereas in MPNSTs from patients with Recklinghausens disease, there was most often a gain in 7q, 8q, 15q, and 17q. The occurrence of gain of both 7p15‐p21 and 17q22‐qter was associated with a statistically significant poor overall survival rate (P = 0.0096). Genes Chromosomes Cancer 25:205–211, 1999.

Amplification of the mdm2 gene, but not expression of splice variants of mdm2 mrna, is associated with prognosis in soft tissue sarcoma

The MDM2 gene encodes a 90‐kDa oncoprotein that is overexpressed in several human carcinomas, osteosarcomas, gliomas and soft tissue sarcomas (STSs). This overexpression is the result of several mechanisms, for example, enhanced transcription or translation, gene amplification and alternative splicing. We found that 19 of 67 (28.4%) STS specimens contained an amplified MDM2 gene. The amplification was more likely to be present in grade 1 tumors than in grade 2 or 3 tumors (58% of grade 1 tumors vs. 15% of grade 2 or 3 tumors, p = 0.001, χ2 test). Furthermore, patients with tumors that contained an amplified MDM2 gene had a survival estimate (87 months) that was longer than that of patients with tumors that lacked an amplified gene (40 months; p = 0.02, log‐rank test). Alternatively and aberrantly spliced MDM2 mRNAs were detected in human STSs by a highly sensitive reverse transcription‐polymerase chain reaction method. Of 71 tumor samples, 38 (54%) showed evidence of the spliced forms, which included MDM2‐A, MDM2‐B and several variants exclusively expressed in STSs. A common feature of all forms was the absence of the MDM2 N‐terminal region, which includes the TP53‐binding region. Furthermore, the presence of the spliced forms was associated with elevated levels of TP53 (p = 0.01, χ2 test). Although the presence of spliced forms was associated with late‐stage tumor phenotypes (p = 0.05, χ2 test), we observed no relationship between the presence of splice variants and patient outcome.