By Dina Lev, MD

Abstract

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.^2-5 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.

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.

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.

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.

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.

V7N1 ESUN Copyright © 2010 Liddy Shriver Sarcoma Initiative.

By Dina Lev, MD and Matt van de Rijn, MD, PhD

Introduction

Liposarcomas account for 15% of adult soft tissue sarcoma (STS); this is a heterogeneous group of tumors that are subdivided into five subgroups based on cytogenic abnormalities.^1,2 Well-differentiated liposarcoma (WD) also known as atypical lipomatous tumor (ALT) and de-differentiated liposarcoma (DD) comprise the vast majority of liposarcoma subtypes (approximately 90%).³ DD liposarcoma was originally described by Evans⁴ in 1979 as a liposarcoma that consists of a combination of ALT and cellular, non lipogenic areas that have significant mitotic activity.

While WD can arise anywhere in the body, DD tumors mainly arise in the retroperitoneum. It is unclear whether WD and DD originate from two different cellular clones or if there is a process of progressive evolution from WD to DD. While this issue is still unresolved, it is unequivocally established that DD constitutes a high grade lesion with increased cellularity that is prone to disseminate and is associated with a much worse prognosis than WD which has minimal metastatic potential; therefore prolonged survival of WD patients is not uncommon.^3,5

Based on the above clinical observations, there is a crucial need to better understand the underlying molecular mechanisms driving these two distinct liposarcoma histological subtypes. Previous studies have demonstrated over-expression of murine double minute (MDM2) and Cyclin-dependent kinase 4 (CDK4) in WD and DD; both genes are associated with tumorigenesis of a variety of cancers.⁶ These findings support the hypothesis that WD and DD liposarcomas might have a common origin; however, they also suggest that additional molecular alternations/dysregulations are necessary to account for the dismal prognosis of DD liposarcoma.

microRNA

Recently, much attention has focused on the impact of microRNAs (miRNAs) on tumorigenesis and cancer progression. miRNAs are single strand RNA molecules of about 22-nucleotides in length that regulate the translation of genes into proteins. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein. Instead, they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA by the nuclear microprocessor complex. Pre-miRNAs are exported to the cytoplasm where they are further processed to mature functional miRNA. The final step in miRNA maturation is the selection and incorporation of one strand of the miRNA into the RNA-induced silencing complex (RISC). The mature molecules are partially complementary to one or more messenger RNA (mRNA), and their binding results in decreased mRNA translation.

Evidence of the miRNA role in development and disease (including cancer) is rapidly accumulating. There is increasing data to suggest that miRNA may act as either a tumor suppressor gene or an oncogene. miRNA deregulation has been identified in a variety of epithelial cancers, where changes in specific miRNAs expression possibly contribute to tumor growth, progression, metastasis, and chemoresistance.⁷ 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; pharmacologic manipulation of miRNA expression has been undertaken in the form of in-vivo miRNA delivery of down-regulated miRNA and "antagomirs" (chemically engineered endogenous miRNA) targeting up-regulated miRNA.⁸

Not much is known about the expression and deregulation of miRNA in soft tissue sarcomas generally and in liposarcoma specifically. The only study published to date evaluating miRNA in soft tissue sarcomas demonstrated distinct miRNA expression signatures among the tumor types studied, suggesting their possible role in sarcomagenesis, and their potential as diagnostic markers or even therapeutic targets (Figure 2).⁹

Only one sample in the evaluated cohort was liposarcoma (DD); therefore, conclusions regarding miRNA expression and deregulation of miRNA in liposarcoma could not be made. Deregulation of miRNA could possibly be an important contributing factor to liposarcoma sarcomagenesis, especially to the process of dedifferentiation. Identifying the miRNA signature of WD and DD liposarcomas could likewise perhaps provide insight into our understanding of the molecular determinants driving these two entities. Furthermore, because these two sarcoma subtypes could represent a disease continuum, such data could possibly enable to identify miRNA deregulations that might accurately predict which WD has the potential for more aggressive dedifferentiation.

Hypothesis

WD and DD liposarcomas harbor unique miRNA expression signatures. The identification of these unique miRNA expression profiles may indicate their role in tumorigenesis and may aid in diagnosis, prognosis and even therapy of liposarcomas.

Experimental Plan

We seek to identify unique miRNA expression profiles of human WD and DD liposarcomas. Using high throughput miRNA arrays, we will compare miRNA expression in frozen human liposarcoma samples of these two distinct subtypes versus matched autologous normal fat. Comprehensive statistical/bioinformatic analysis will be conducted to identify miRNAs that are selectively deregulated in either of these liposarcoma subtypes and to establish the differences in miRNA expression profiles that distinguish between them. Furthermore, we will evaluate the possible differences between miRNA in pure WD tumors versus that in the WD portions of DD liposarcoma, with the hope of identifying miRNA expression patterns predicting sarcomagenic progression. Identified miRNA patterns will be further validated by quantitative RT-PCR examination of a large cohort of paraffin-embedded archival liposarcoma specimens. 

V5N4 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative.

Grant Funding

The Liddy Shriver Sarcoma Initiative awarded this $50,000 grant in August 2008. The study was made possible by a generous gift from the Keating family in honor of James Keating, and by a generous gift from Dr. Laura Somerville.