We have demonstrated that sequencing-based miRNA expression profiles discriminate normal fat, WDLS, and DDLS. MicroRNA-143, which is abundant in normal adipose tissue, is underexpressed in WDLS, and its expression decreases further as tumors progress to DDLS. These findings imply that miR-143 underexpression is an early event in liposarcomagenesis, but that further loss may play a role in dedifferentiation. Esau et al. previously demonstrated that miR-143 levels increase in differentiating adipocytes and that miR-143 is required for differentiation (21
). We found as well that miR-143 levels increase in differentiating adipocytes; however, re-expression of miR-143 is insufficient to restore differentiation in the cellular context of DDLS. Therefore, while miR-143 loss may produce effects through inhibition of differentiation, miR-143 restoration alone is unable to reverse this phenotype in DDLS cells.
Re-expression of miR-143 but not its co-cistronic partner miR-145 inhibits DDLS cell proliferation and induces apoptosis. MiR-143 re-expression also decreases entry into S phase and inhibits mitosis. The effects of miR-143 on proliferation, apoptosis, and cell cycle progression may be explained by a gene network targeted by miR-143, including direct miR-143 targets BCL2, TOP2A, and PRC1 and the indirect target PLK1. This network includes genes involved not only in cell proliferation and apoptosis, but also in cytokinesis. Pharmacologic inhibition of PLK1, a regulator of cytokinesis, induces apoptosis and cell cycle arrest in DDLS cells.
Prior work has implicated miR-143 as a tumor suppressor in other cancers. In bladder cancer, miR-143 inhibits cancer growth through antagonizing the expression of RAS (28
). Both miR-143 and miR-145 are downregulated in colon cancer and in B-cell malignancies, such as chronic lymphocytic leukemia and Burkitt's lymphoma (29
). MiR-143 in particular appears to be a tumor suppressor in colorectal cancer, acting through downregulation of KRAS, a mediator of the MAPK cascade (30
). In these other cancer types, the main effect of miR-143 re-expression is inhibition of proliferation without induction of apoptosis. In contrast, in liposarcoma cells we detect significant induction of apoptosis after only 48 hours of miR-143 re-expression.
We have shown that miR-143 downregulates BCL2, TOP2A
, and PRC1
in DDLS cells. BCL2
encodes a mitochondrial membrane protein that blocks apoptosis and has been implicated in multiple human cancers. It was recently identified as a direct target of miR-143 in osteosarcoma (31
) and bears at least two predicted miR-143 target sites (Supplemental Table 3
). The repression of BCL2
may contribute to miR-143's pro-apoptotic properties, but, given miR-143's lack of significant effect on BCL2
in one of the two DDLS cell lines, this may not apply to all DDLS tumors.
TOP2A (topoisomerase 2a) is an enzyme controlling the topologic state of DNA and involved in transcription and DNA replication. It is upregulated in multiple human cancers such as breast and prostate cancer, and its expression is associated with androgen resistance and decreased survival in prostate cancer. Our group has found that TOP2A
expression is upregulated 43-fold in DDLS relative to normal fat (Supplemental Table 4
). Furthermore, TOP2A
upregulation in liposarcomas was associated with decreased distant recurrence–free survival. shRNA knockdown of TOP2A
causes induction of apoptosis and decreased invasive ability of DDLS cells (32
), underscoring its importance in this disease.
To date, the regulation of TOP2A in DDLS and other tumors has been poorly defined. Our study provides the first evidence that miR-143 regulates TOP2A and that this regulation may partially explain miR-143's antiproliferative and pro-apoptotic effects. In addition, miR-143's regulation of TOP2A, a key enzyme of DNA replication, may explain the decreased progression into S phase after miR-143 restoration.
TOP2A is a target of anthracycline-based chemotherapeutic agents, such as doxorubicin, which alter TOP2A catalysis, generating high levels of DNA breaks that then trigger cell death pathways. These agents, however, are not completely TOP2A-specific, and their use is limited by high toxicity and poor efficacy in DDLS. The present results suggest that rational screening for more potent and specific TOP2A-targeted agents may lead to more effective therapy for patients with liposarcoma.
Protein regulator of cytokinesis 1 (PRC1) and polo-like kinase 1 (PLK1) are crucial regulators of cytokinesis. PRC1, a docking partner of PLK1, recruits PLK1 to the central spindle during anaphase (22
). Suppression of PRC1
) or chemical inhibition of PLK1 (35
) results in mitotic failure, and both proteins are required for proper cytokinesis. Our group has previously found that PRC1
is upregulated 24-fold in DDLS relative to normal fat (13
). We demonstrated here that miR-143 negatively regulates PRC1
expression, that miR-143 restoration inhibits mitosis in DDLS cells, and that PLK1 inhibition via drug treatment leads to mitotic arrest and cell death. These observations, together with the microarray and network analysis implicating miR-143's regulation of other genes involved in cytokinesis (ECT2
, and CDC25B
), suggest that miR-143 plays an important role in regulating cytokinesis in liposarcoma cells. Furthermore, they imply that PLK1 inhibitors may have potential for the treatment of DDLS.
Key to our study has been elucidation of the complex regulatory network of miR-143. Consistent with the observation that microRNAs target a multitude of genes, each of which has a small but significant effect on the overall phenotype conferred by the microRNA, we identified a strongly connected network regulated by miR-143. Interestingly, expression levels of 10 of the 24 genes in the network are associated with decreased distant-recurrence–free survival (32
) (Supplemental Table 4
). In addition, 9 of these 10 genes are upregulated significantly in DDLS relative to normal fat (13
). The 24-gene network controls several important cellular functions ultimately converging in cell growth and survival. While agents targeting individual miR-143 targets may provide useful therapy for DDLS, our results suggest therapy targeting the entire dysregulated gene network via delivery of miR-143 to DDLS tumors may provide more effective therapy.