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

Abstract

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=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.

Main

It has been proposed that several tumours including soft-tissue sarcomas (STS) may originate from stem cells. Stem cells and tumour stem cells share several similarities, that is, the ability of self-renewal without loss of proliferation capacity and a relative resistance to drugs. Dysregulation of stem cell self-renewal may be a prerequisite for the development of cancer (Soltysova et al., 2005).

Previously, we evaluated the mRNA expression of two genes, survivin and hTERT, in soft-tissue sarcoma. Both genes are predominantly expressed in stem cells and tumour cells but hardly at all, if any, in normal tissues. In a multivariate Cox's proportional hazards regression model, we found that increased expression of hTERT (P=0.034; relative risk (RR)=5.8, 95% confidence interval (CI) 1.1–29.6) and the coexpression of survivin and hTERT (P=0.0004; RR=20.1, 95% CI 3.8–106.4) were significantly correlated with a poor survival for STS patients (Würl et al., 2002). However, additional studies to evaluate expression of stem cell-associated genes in STS have yet to be performed. In this study, we investigated the transcript level of the gene Hiwi (Piwi-1 related), which encodes a protein responsible for stem cell self-renewal. The level of Hiwi transcript was compared to patient survival to ascertain its relationship to prognosis.

We analysed 65 frozen primary tumour samples from 65 adult STS patients (before an adjuvant treatment) by real-time quantitative PCR for Hiwi mRNA expression. All patients gave written informed consent (Institute of Pathology, University of Halle, Germany and Department of Surgery 1, University of Leipzig, Germany). The mRNA expression of Hiwi was quantified by a commercially available TaqMan gene expression assay (Assay-ID: Hs01041737_m1; Applied Biosystems, Foster City, CA, USA) and standardized to the transcript level of hypoxanthin-phospho-ribosyl-transferase (HPRT), using the TaqMan gene expression assay (Assay-ID: Hs99999909_m1, Applied Biosystems).

A median ratio of Hiwi/HPRT transcript level of 0.056 (0–17.1) attogram (ag) Hiwi mRNA/ag HPRT mRNA could be detected in the 65 STS samples and a median ratio of 0.12 (0–1.9) was found in tumour adjacent tissue (muscle, n=27). Relative expression levels of Hiwi were defined as high, medium, and low when the relative values were >1.5 ag Hiwi mRNA/ag HPRT mRNA, >0.05 to 1.5 ag Hiwi mRNA/ag HPRT mRNA and 0.05 ag Hiwi mRNA/ag HPRT mRNA, respectively (Table 1). STS of 32 patient tumours expressed a low Hiwi relative expression level. Tumours of 20 patients expressed a medium level and 13 patient tumours expressed high-level Hiwi mRNA. We performed multivariate analysis according to Cox's proportional hazards regression model adjusted to prognostic relevant factors (tumour stage, tumour localization, tumour entity, tumour resection and expression of survivin and hTERT). In this analysis, we found that an increased expression of Hiwi mRNA is correlated with a 4.6-fold (1.3–16.1) increased risk of tumour-related death for STS patients (P=0.017; Figure 1). A low expression of Hiwi transcript is correlated with a 2.4-fold (0.7–8.0) increased risk, but this effect was not significant (P=0.17; Figure 1). These results suggest that a medium expression level of Hiwi may represent the normal physiological status in a mesenchymal cell. However, dysregulation of Hiwi mRNA transcription, that is, both a high and a low expression of Hiwi mRNA could affect prognosis.

Table 1 Histopathological and clinical data
Figure 1
figure1

Cox's proportional hazards regression model. Adjusted to tumour stage, tumour localization, tumour entity, tumour resection and expression of Hiwi for 65 patients with STS in 100 months. Cutoffs for expression of Hiwi were >1.5 ag (high), >0.05 to 1.5 ag (medium) and 0.05 ag mRNA (low) standardized to ag HPRT expression.

Hiwi is a member of the Piwi gene family named after the first member Piwi identified in Drosophila melanogaster (Cox et al., 2000). The Piwi family represents the first class of genes known to be required for stem cell self-renewal in diverse organisms such as jellyfish, Caenorhabditis elegans, D. melanogaster, zebrafish, mouse and humans (reviewed by Liu et al., 2006). Hiwi mRNA is present in human CD34(+) haematopoietic progenitor cells, but not in more differentiated cell populations (Sharma et al., 2001). In addition to its function in stem cell renewal, an overexpression of Piwi causes an increase both in the number of germline stem cells and the rate of their divisions in D. melanogaster (Cox et al., 2000). Consistent with this finding, overexpression of Hiwi in male germline cells correlates with the occurrence of seminomas, that is, a type of testicular germ cell tumours (Qiao et al., 2002). Recently, Liu et al. (2006) showed that the percentage of cells that expressed Hiwi increased from 10% in normal gastric tissues to 76% in gastric cancer. Furthermore, the expression of Hiwi was associated with proliferation of cancer cells (Liu et al., 2006).

How could an overexpression of Hiwi affect tumour progression? All members of the Piwi gene family possess a so called PIWI domain of about 300 amino acids (Cerutti et al., 2000), which has a structural homology to RNase H endonuclease (Parker et al., 2004). Proteins with Piwi domains have been described as components of ribonucleoprotein complexes that act in the microRNA/RNA interference pathway of gene silencing (Fetzer et al., 2002; Lingel and Sattler, 2005). Recently, two groups could show that members of the Piwi gene family are associated with a new class of small RNAs so-called ‘piwi-interacting RNAs’ (piRNAs) in mammalian spermatogenesis (Aravin et al., 2006; Girard et al., 2006). The authors discuss that the complex of germline-specific Piwi proteins and piRNAs could have regulatory functions in mammalian spermatogenesis by timing of meiotic and post-meiotic events through transcriptional and translational repression or by supporting chromosome homology searching and chromosome pairing (Aravin et al., 2006; Girard et al., 2006). The Piwi protein Hiwi could play a role in the balance between stem cell self-renewal and stem cell division in association with small RNAs' pathway. A disturbance in this balance may have strong impact on tumour progression.

Until now, a correlation between Hiwi transcript expression and prognosis has not been described. In this study, we make the novel observation that an increased expression of Hiwi correlates with a poor outlook for STS patients. In conclusion, our results indicate that Hiwi gene expression may be used as predictor of survival for patients with STS, and potentially other cancers.

Abbreviations

ag:

attogram

CI:

confidence interval

RR:

relative risk

STS:

soft-tissue sarcoma(s)

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft Project No. TA 145/8-19 and a grant from the Deutsche Krebshilfe Project No.10-2130-Ta2. We thank Deanna Naeve and Dr Clayton Naeve from St Jude Children's Research Hospital, Memphis, TN, USA for continuous support and helpful discussions. The funding source had no role in study design, data collection, data analysis, data interpretation or writing of the report.

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Taubert, H., Greither, T., Kaushal, D. et al. Expression of the stem cell self-renewal gene Hiwi and risk of tumour-related death in patients with soft-tissue sarcoma. Oncogene 26, 1098–1100 (2007). https://doi.org/10.1038/sj.onc.1209880

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Keywords

  • Hiwi
  • stem cell self-renewal
  • soft-tissue sarcoma
  • prognosis
  • Piwi domain
  • mRNA expression

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