Elsevier

Gynecologic Oncology

Volume 140, Issue 3, March 2016, Pages 512-517
Gynecologic Oncology

Uterine leiomyosarcoma and endometrial stromal sarcoma have unique miRNA signatures

https://doi.org/10.1016/j.ygyno.2016.01.001Get rights and content

Highlights

  • microRNA array analysis differentiates uterine endometrial stromal sarcoma from leiomyosarcoma.

  • microRNAs are predominantly underexpressed in metastatic compared to primary leiomyosarcoma.

  • Frizzled-6 silencing suppresses invasion in leiomyosarcoma cells in vitro.

Abstract

Objective

To compare the microRNA (miRNA) profiles of uterine endometrial stromal sarcoma (ESS) and leiomyosarcoma (LMS), and to compare the miRNA signatures of primary and metastatic uterine LMS.

Methods

Eight primary LMS, 9 primary ESS and 8 metastatic LMS were analyzed for miRNA profiles using TaqMan Human miRNA Array Cards. Findings for 20 differentially expressed miRNAs were validated in a series of 44 uterine sarcomas (9 primary uterine ESS, 17 primary uterine LMS, 18 metastatic LMS) using qPCR. Frizzled-6 protein expression was analyzed in 30 LMS (15 primary, 15 metastases). Frizzled-6 was silenced in SK-LMS-1 uterine LMS cells using siRNA and the effect on invasion, wound healing and matrix metalloproteinase-2 (MMP2) activity was assessed.

Results

Ninety-four miRNAs were significantly differentially expressed in ESS and LMS, of which 76 were overexpressed in ESS and 18 overexpressed in LMS. Forty-nine miRNAs were differentially expressed in primary and metastatic LMS, of which 45 were overexpressed in primary LMS and 4 in metastases. Differential expression was confirmed for 10/20 miRNA analyzed using qPCR. Frizzled-6 silencing in SK-LMS-1 cells significantly inhibited cellular invasion, wound healing and MMP-2 activity.

Conclusions

Differential miRNA signatures of ESS and LMS provide novel data regarding transcriptional regulation in these cancers, based on which new potential diagnostic markers, prognostic biomarkers and therapeutic targets may be explored. Differences in miRNA profiles of primary and metastatic LMS may improve our understanding of disease progression in this aggressive malignancy.

Introduction

Uterine sarcomas are rare tumors, comprising 7% of all soft tissue sarcomas and about 3% of uterine malignancies [1], [2], [3]. Leiomyosarcoma (LMS) and endometrial stromal sarcoma (ESS) are the most common histological types [2], [3]. Adenosarcoma and carcinosarcoma are both mixed epithelial–mesenchymal tumors, but only the former have a true sarcomatous component, whereas carcinosarcomas are currently regarded as metaplastic carcinomas. ESS has been previously classified as low-grade or high-grade, later regarded as a single entity, and recently re-divided into low-grade and high-grade categories, although the latter group constitutes rare tumors [4].

MicroRNAs (miRNAs) are small (19–25 nucleotides), non-coding RNAs that post-transcriptionally regulate gene expression [5]. miRNAs are synthesized as a long double-stranded precursor called pri-miRNA by DNA polymerase II in the nucleus. Pri-miRNA is cleaved at specific sites by the RNAse Drosha inside the nucleus, producing a pre-miRNA which is exported to the cytoplasm by the exportin 5 protein, where it is processed by Dicer into mature miRNA. Mature miRNAs are subsequently activated through binding to the RNA-induced silencing complex (RISC) [6], [7]. Through the RISC, miRNAs can regulate their targets, mediating translational repression or degradation. The sequence at the 5′ end of the mature miRNA, called the “seed region”, binds its complementary sequence within the 3′ untranslated regions (UTR) of the target mRNA [8]. Perfect or near-perfect complementarity between the miRNA and its mRNA target results in mRNA degradation, whereas lesser complementarity leads to translational inhibition.

Data regarding the miRNA profile of uterine sarcomas is limited to date. An inverse association was reported for the miRNA Let-7 and its target high-mobility group AT-hook-2 (HMGA2) in uterine LMS [9]. miRNA profiles which differentiate uterine LMS from leiomyoma [10] or from different types of leiomyoma as well as smooth muscle tumors of uncertain malignant potential (STUMP) [11] have been described. In another report, the miRNA profiles of uterine sarcomas and carcinosarcomas were compared to those of patient-matched benign tissue [12].

Our group has previously reported on gene expression profiles which differentiate LMS from ESS [13] and primary LMS from metastatic LMS [14]. Similar data with respect to miRNA profiles are unavailable to date to the best of our knowledge. The present study compared the miRNA profiles of primary ESS, primary LMS and metastatic LMS.

Section snippets

Patients and material

Specimens consisted of 44 uterine sarcomas, including 9 primary uterine ESS, 17 primary uterine LMS and 18 metastatic LMS, submitted for routine diagnostic purposes to the Department of Pathology at the Norwegian Radium Hospital during the period 1993–2009. Tumors were snap-frozen and kept at − 70 °C. Frozen sections were evaluated for the presence of a > 80% tumor component and absence of necrosis. Diagnoses were established by an experienced gynecologic pathologist (BD) based on morphology and

Results

Forty-nine miRNAs were found to be differentially expressed between primary and. metastatic LMS, of which 4 were overexpressed and 45 underexpressed in the metastatic lesions. Comparative analysis of primary LMS and ESS identified 94 miRNAs that were significantly differentially expressed between these two sarcoma types, including 76 overexpressed in ESS and 18 overexpressed in LMS (p < 0.05; Supplementary Table 1 and Supplementary Figs. 1-A, -B).

Among the 20 miRNAs found to be most significantly

Discussion

ESS and LMS are both rare sarcomas affecting the uterine corpus. However, they differ considerably at the genotypic and phenotypic levels, as well as their clinical behavior. Our understanding of differences in the molecular drivers of each of these cancers is limited by the paucity of comparative molecular analyses of ESS and LMS. Beyond scientific interest, such studies may improve and expand the very limited panel available for differentiating these tumors in routine surgical pathology,

Financial acknowledgment

This work was supported by a grant from the National Sarcoma Foundation at the Norwegian Radium Hospital.

Conflict of interest statement

The authors declare that they have no competing interests.

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    1

    R.R. is affiliated with the David R. Bloom Center for Pharmacy and the Adolf and Klara Brettler Center for Research in Molecular Pharmacology and Therapeutics at The Hebrew University of Jerusalem, Israel.

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