High throughput miRNA expression profiling for well differentiated and de-differentiated liposarcoma: a progress report
An ESUN Article
Editor's Note: This article is a follow-up to the study proposal entitled: "High throughput miRNA expression profiling for well differentiated and de-differentiated liposarcoma," which was published in the August 2008 issue of ESUN. This study was funded by a grant from the Liddy Shriver Sarcoma Initiative.
Introduction
Liposarcomas (LPS) account for approximately 15% of adult soft tissue sarcoma (STS); this is a heterogeneous group of tumors composed of several histologic subtypes. Well differentiated (WDLPS), also known as atypical lipomatous tumor (ALT), and dedifferentiated liposarcomas (DDLPS) are the most common subtypes. Dedifferentiated LPS was originally described by Evans as a liposarcoma that consists of a combination of atypical lipomatous tumor and cellular elements; the latter components can have significant mitotic activity (1). While WDLPS can arise anywhere in the body, DD tumors mainly arise in the retroperitoneum (RPS). While still debated, several lines of evidence suggest that WDLPS and DDLPS represent a disease continuum. Most importantly, WDLPS and DDLPS exhibit different clinical behaviors and outcomes, with DDLPS having a significantly worse prognosis (2, 3, 4, 5). Long term survival of patients with WDLPS is common in that these tumors lack metastatic potential, whereas DDLPS are markedly aggressive and distant metastases and/or multi- focal recurrence followed by death constitutes a typical clinical course. Based on these clinical observations there is a crucial need to better understand the underlying molecular mechanisms driving these two distinct LPS subtypes. Cytogenic analysis and comparative genomic hybridization (CGH) studies have demonstrated that both WDLPS and DDLPS are characterized by supernumerary ring chromosomes or giant marker chromosomes and12q13–15 region amplifications, resulting in MDM2 and CDK4 overexpression. These findings support the hypothesis that WDLPS and DDLPS might have a common origin, however they also suggest that additional molecular alterations/dysregulations are necessary to account for the dismal prognosis of DDLPS.
Well-Differentiated and De-Differentiated Liposarcomas: A comprehensive article summarizing the differences between WD and DD liposarcomas appears in the June 2008 issue of ESUN.
Recently, much attention has focused on the impact of microRNAs (miRNAs) on tumorigenesis and cancer progression. miRNAs are approximately 22-nucleotide non-coding RNAs that participate in post transcriptional gene expression regulation through mRNA degradation, translational inhibition or chromatin-based silencing mechanisms. Evidence of miRNAs role in development and disease (including cancer) is rapidly accumulating; there is increasing data to suggest that miRNAs may act as either tumor suppressors or oncogenes. miRNA deregulation has been identified in a variety of epithelial origin cancers, where changes in specific miRNAs expression possibly contribute to tumor growth, progression, metastasis, and chemoresistance (6). Furthermore, several recent studies have highlighted the potential of miRNA profiles for diagnosis and prognosis. Stemming from these initial reports, ongoing investigations are evaluating the potential usefulness of miRNA-based therapy in cancer, and point to a potential role of sensitize cancer cells following chemo- and radiotherapy. Pharmacologic manipulation of microRNA expression has been undertaken in the form of in vivo miRNA delivery of downregulated miRNAs and "antagomirs" targeting upregulated miRNAs (7).
Not much is known about the expression and deregulation of miRNA in STS generally and in LPS specifically. In a recent study using a microarray approach, 27 STS of seven different histological subtypes were profiled for miRNA expression (8). The results demonstrated that miRNA expression signatures were clearly distinct among the tumor types studied, suggesting their possible role in sarcomagenesis, and their potential as diagnostic markers or even therapeutic targets. Only one sample in the evaluated cohort was LPS (DDLPS); consequently, no major conclusions regarding the expression and regulation of miRNA in LPS could be made. However, the feasibility of such studies is suggested by these findings. Dysregulation of miRNA could possibly be an important contributing factor to LPS sarcomagenesis, especially to the process of dedifferentiation. Our studies, supported by the Liddy Shriver Sarcoma Initiative, aimed to identify unique miRNA expression profiles of WDLPS and DDLPS with the hope that such signatures could provide insights into our understanding of the molecular determinants driving these two entities.
Results
To gain insight into the miRNA profiles of LPS we have utilized a panel of frozen human LPS specimens (n=17) and corresponding normal fat (n=8) from which high quality and integrity total RNA was extracted. miRNA profiling was conducted using miRCURY LNA™ microRNA Arrays (Exiqon, Denmark). Data analysis identified 39 differentially expressed miRNA in LPS specimens as compared to normal fat (p<0.001; Figure 1); four miRNAs were found to be over-expressed in LPS and 35 were down-regulated. Several miRNAs were selected for array validation and were shown to be down regulated in an independent cohort of LPS specimens as compared to normal fat using qRT-PCR.
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Importantly, comparison of WDLPS and DDLPS miRNA profiles identified 45 differentially expressed miRNAs (p<0.05) of which 11 were found to be over-expressed in DDLPS and 34 were found to be down-regulated (Figure 2). As per above, several miRNAs were further selected to validate the array results via qRT-PCR demonstrating differential expression in WDLPS vs. DDLPS in an independent tissue cohort.
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Preliminary results presented above are currently being expanded and the role of the identified miRNAs as prognostic markers and therapeutic targets for LPS is undergoing evaluation. One major obstacle to the feasibility of such studies is the lack of human WDLPS and DDLPS cell lines. To that end we have been isolating primary LPS cultures from fresh surgically resected tumors. These efforts have resulted in the acquisition of several LPS cell strains (WDLPS and DDLPS) which have now been growing continuously for more than 20 passages in culture (Figure 3). These cells have been demonstrated to retain the 12q15 amplification as was revealed by interphase FISH and were extensively characterized. Differential expression of selected miRNAs identified above was further confirmed in these cells demonstrating the potential use of this model for future experiments.
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Summary and Future Directions
The role of miRNA dysregulation in tumorigenesis and cancer progression has recently become established. However, miRNA expression and function in LPS is unknown. Under the aegis of the proposed studies that incorporate a systematic high throughput approach, we sought to identify subtype-specific differentially expressed miRNAs that may be involved in LPS progression. We hope to continue by evaluating the impact of candidate miRNAs on LPS tumorigenesis and progression in vitro using a panel of human LPS cells. Additionally, we will study the utility of these miRNAs as therapeutic targets in vivo using relevant animal models. We will prospectively test the utility of the unique miRNA signatures in the diagnosis of human LPS, and most importantly, determine their value to identify those WDLPS with the greatest potential to undergo DD progression. Such a molecular tool would be of major significance in therapeutic decision making. Future studies will hopefully demonstrate the potential of the identified miRNAs as diagnostic markers or perhaps even new therapeutic targets for LPS.
References
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2. Lahat G, et al. Resectable well differentiated versus dedifferentiated liposarcomas: Two different diseases possibly requiring different treatment approaches. Ann Surg Oncol 2008; 15:1585-93.
3. Linehan DC, et al. Influence of biologic factors and anatomic site in completely resected liposarcoma. J Clin Oncol 2000; 18:1637-1643.
4. Gronchi A, et al. Retroperitoneal soft tissue sarcomas: patterns of recurrence in 167 patients treated at a single institution. Cancer. 2004; 100:2448-2455.
5. Singer S, et al. Histologic subtype and margin of resection predict pattern of recurrence and survival for retroperitoneal liposarcoma. Ann Surg 2003; 238:358–370.
6. Kent OA, et al. A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Oncogene 2006; 25:6188-6196.
7. Krützfeldt J, et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 2005; 438:685-9
8. Subramanian S, et al. MicroRNA expression signature of human sarcomas. Oncogene 2008; 17: 2015-2026.
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