High throughput miRNA expression profiling for well differentiated and de-differentiated liposarcoma

Dina Lev, MD

Assistant Professor of Cancer Biology

The University of Texas M D Anderson Cancer Center

Houston, Texas

and

Matt van de Rijn, MD, PhD

Professor of Pathology

Stanford University School of Medicine

Stanford, California

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 morphology and cytogenic abnormalities (Refs. 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%; Ref. 3). DD liposarcoma was originally described by Evans (Ref. 4) 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 (Refs. 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 (Ref. 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.

Figure 1: Diagram depicting miRNA processing

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 (Ref. 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 oligonucleotides which are utilized to silence endogenous miRNA) targeting up-regulated miRNA (Ref. 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 (Ref. 9; Figure 2, below).

Figure 2: Unsupervised hierarchical analysis of 27 sarcomas and two normal skeletal muscle samples. Each row represents the relative levels of expression for a single miRNA and each column shows the expression levels for a single sample. The red or green color indicates relatively high or low expression, respectively, while gray indicates absent data points (SS-synovial sarcoma; ERMS-embryonal rhabdomyosarcoma (RMS); ARMS-alveolar RMS; PRMS-pleomorphic RMS; SKM-skeletal muscle; GIST-gastrointestinal stromal tumor; LMS-leiomyosarcoma; DDLPS-dedifferentiated liposarcoma. [Figure 2 contributed by S. Subramanian, unpublished results.]

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.  

References

1. Dei Tos AP. Liposarcoma: new entities and evolving concepts. Ann Diagn Pathol 4:252-266, 2000

2. Mack TM. Sarcomas and other malignancies of soft tissue, retroperitoneum, peritoneum, pleura, heart, Mediastinum, and spleen. Cancer 75:211-244, 1995

3. Singer S, Antonescu CR, Riedel E, et al: Histologic subtype and margin of resection predict pattern of recurrence and survival for retroperitoneal liposarcoma. Ann Surg 238:358-370, 2003

4. Evans HL, Soule EH, Winkelmann RK: Atypical lipoma, atypical intramuscular lipoma, and well differentiated retroperitoneal liposarcoma: a reappraisal of 30 cases formerly classified as well differentiated liposarcoma. Cancer 43:574-84, 1979

5. Lahat G, Anaya DA, Wang X, et al: Resectable well-differentiated versus dedifferentiated liposarcomas: two different diseases possibly requiring different treatment approaches. Ann Surg Oncol 15:1585-93, 2008

6. Segura-Sánchez J, González-Cámpora R, Pareja-Megia MJ, et al. Chromosome-12 copy number alterations and MDM2, CDK4 and TP53 expression in soft tissue liposarcoma. Anticancer Res 26:4937-42, 2006

7. Kent OA, Mendell JT. A small piece in the cancer puzzle: microRNAs as tumor suppressors and oncogenes. Oncogene 25:6188-96, 2006

8. Krützfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438:685-9, 2005

9. Subramanian S, Lui WO, Lee CH, Espinosa I, Nielsen TO, Heinrich MC, Corless CL, Fire AZ, van de Rijn M. MicroRNA expression signature of human sarcomas. Oncogene 27:2015-26, 2008

V5N4 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative.