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.

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 5 66) and HDMX-S mRNA expression (n 5 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 Cox's 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 5 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. ' 2005 Wiley-Liss, Inc.Key words: HDMX; amplification; overexpression; oncogene; prognosis; alternative splicing; cancer Recently, a protein related to the product of the murine double minute gene 2 (MDM2), designated as HDMX, has been identified, and the HDMX gene was mapped to chromosome region 1q32. 1,2 It has been shown that HDMX (also referred to as MDM4 or MDMX in the mouse) is able to bind to p53, thereby inhibiting its activity. However, it fails to induce the nuclear export of p53 or its proteosomal degradation. 3 Although HDMX bears resemblance to MDM2, it has some distinct features. For example, whereas expression of MDM2 can be induced by DNA damaging agents, HDMX levels are not. 1 This suggests that the HDMX promoter, unlike the P2-promoter of the MDM2 gene, is not transcriptionally transactivated by p53. Data from gene knockout experiments indicate that embryonic lethality of both MDMX 2/2 and MDM2 2/2 mice can be rescued by the loss of p53. 4,5 Therefore, it seems likely that MDM2 and MDMX act in nonoverlapping pathways to regulate p53. 5 However, data also show that HDMX actively regulates p53 activity through its interaction with MDM2, 6 suggesting that HDMX/MDMX is another important regulator of the p53-MDM2 network (reviewed in Michael and Oren 7 ). These data raise the possibility that increased levels of HDMX result in the inactivation of p53 that may contribute to tumor development. In fact, several studies have revealed that the HDMX gene is a target of amplification at the 1q32 gene locus in malignant gliomas that have no p53 mutations or MDM2 amplification. 8,9 In addition, the HDMX gene has been shown to be amplified in breast canc...

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.

The aim of this study was to investigate the level and the prognostic value of the expression of different survivin transcript variants -survivin, survivin-DEx3 and survivin-2B -in tumours of 76 soft tissue sarcoma (STS) patients. The expression of survivin transcript variants in STS tissue samples and in 12 nonmalignant control tissues was analysed by quantitative RT-PCRs. Expression levels of all survivin transcript variants were strongly elevated in STS compared to normal tissues. A positive correlation between expression of splice variants and tumour stage was found (P ¼ 0.02; v 2 test). The multivariate Cox's proportional hazards regression model revealed a 7.3-fold increased risk of tumour-related death for patients with survivin-DEx3 overexpressing tumours (P ¼ 0.007). The effect of surivivin (wildtype variant) and survivin-2B was less pronounced but still significant (2.2-and 1.9-fold, resp., Po0.05 each). Our results show for the first time that mRNA expression of survivin-variants is significantly correlated to a poor prognosis for STS patients, and we suggest expression of survivin splice variants together with tumour stage as independent predictor of survival.

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