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 Cox's proportional hazards regression model, we found that an increased expression of Hiwi mRNA is a significant negative prognostic factor for patients with STS (P ¼ 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 ¼ 0.17). Altogether, high-level expression of Hiwi mRNA identifies STS patients at high risk of tumourrelated death. This is the first report showing a correlation between expression of a gene involved in stem cell selfrenewal and prognosis of cancer patients.

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...

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BackgroundHypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1α inhibition on radioresistance of malignant glioma.MethodsIn this study, we investigated the effects of the inhibition of HIF-1α on cell survival and radiosensitivity in U251MG and U343MG glioma cells, using two different strategies. HIF-1α inhibition was achieved by siRNA targeting of HIF-1α or via chetomin, a disruptor of interactions between HIF-1α and p300. The inhibition of the HIF-1 pathway was monitored by quantitative real-time PCR and Western blot analyses of the expression levels of HIF-1α and CA9. CA9 expression was investigated as a potential indicator of the efficacy of HIF-1 inhibition and the resulting radiosensitivity of malignant glioma cell lines was determined by clonogenic assay after irradiation under normoxic (2-10 Gy) or hypoxic (2-15 Gy) conditions.ResultsAlthough siRNA and chetomin show distinct modes of action, both attenuated the hypoxia-induced radioresistance of malignant glioma cell lines U251MG (DMF10: 1.35 and 1.18) and U343MG (DMF10: 1.78 and 1.48). However, siRNA and chetomin showed diverse effects on radiosensitivity under normoxic conditions in U251MG (DMF10: 0.86 and 1.35) and U343MG (DMF10: 1.33 and 1.02) cells.ConclusionsResults from this in vitro study suggest that inhibition of HIF-1α is a promising strategy to sensitize human malignant gliomas to radiotherapy and that CA9 could serve as an indicator of effective HIF-1-related radiosensitization.

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 Recklinghausen's 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 Recklinghausen's 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. © 1999 Wiley‐Liss, Inc.

Poor oxygenation of solid tumors is a major indicator of adverse prognosis after standard treatment, e.g. radiotherapy. This observation founded on intratumoral pO(2) electrode measurements has been supported more recently by studies of injected hypoxia markers (pimonidazole, EF5) or hypoxia-related proteins (hypoxia-inducible factor-1alpha, carbonic anhydrase IX) detected immunohistochemically. Alternative approaches include imaging of tumor hypoxia by nuclear medicine studies and the measurement of hypoxia-related proteins (osteopontin) in patient plasma. Low oxygen levels as found in tumors are rarely observed in normal tissues. The presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but as an opportunity for tumor-specific treatment. Classic approaches to normalize tumor oxygenation involve the breathing of modified gas mixtures and pharmacologic modification of blood flow as in the "accelerated radiotherapy, carbogen, nicotinamide" (ARCON) scheme. Specific killing of hypoxic tumor cells can potentially be achieved by hypoxia-selective cytotoxins (model substance tirapazamine), which has shown promise in head and neck cancer. Direct targeting of hypoxia-related molecules such as hypoxia-inducible factor-1alpha, the central regulator of the hypoxic response in tumor cells, is an attractive approach currently tested in preclinical models. For clinical applications, the appropriate combination of hypoxia detection for patient selection with a hypoxia-specific treatment is essential. A therapeutic benefit has been suggested for the selection of patients by plasma osteopontin level and treatment with the hypoxic radiosensitizer nimorazole in addition to radiotherapy, for selection by F-misonidazole positron-emission tomography (PET) and treatment with tirapazamine in addition to chemoradiation and for selection by pimonidazole immunohistochemistry and ARCON treatment, all in head and neck cancer.

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 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. © 2001 Wiley‐Liss, Inc.