Original ArticleA miR-135b-TAZ positive feedback loop promotes epithelial–mesenchymal transition (EMT) and tumorigenesis in osteosarcoma
Introduction
Osteosarcoma (OS) is the most common type of primary malignant bone tumor that mainly occurs in children and adolescents and has a high rate of local invasion and metastasis [1]. Chemotherapy and surgical intervention can increase the 5-year survival rate to 60%–70% [2]; however, the rate is only 30% in patients with a metastatic tumor [3], [4]. It is therefore important to clarify the molecular mechanisms underlying OS metastasis and identify new therapeutic targets in order to improve patient prognosis.
Recently, a newly discovered and evolutionarily and functionally conserved signaling pathway, Hippo pathway, has been shown to play a critical role in controlling organ size by regulating both cell proliferation and apoptosis [5]. The core Hippo signaling complex in mammals is composed of two kinases, Mst1/2 (mammalian sterile twenty-like) and Lats1/2 (large tumor suppressor). Mst1/2 forms a complex with the adaptor protein Sav1 to phosphorylate and activate Lats1/2. The activated Lats1/2, in association with the tumor suppressor Mob1, then phosphorylates and inhibits transcriptional coactivators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) [6]. TAZ and YAP do not directly bind to DNA but can be recruited to their target promoters through binding to the TEA domain (TEAD/TEF) transcription factors [7]. In mammals, there are four highly conserved TEAD/TEF transcription factors (TEAD1, TEAD2, TEAD3, TEAD4). These proteins share in common the TEA DNA binding domain, and are referred to as the TEAD proteins, and require transcriptional coactivators for transcription activation. Three broad groups of candidates for these coactivators have been identified and classified including YAP1, TAZ, vgll proteins, and p160 family of nuclear receptor coactivators [8]. With the help of coactivators, the TEAD family plays a crucial role in some physiological processes as well as in human cancers.
Tumor metastasis begins with epithelial–mesenchymal transition (EMT), a process in which epithelial cells assume a mesenchymal character and become migratory and invasive [9], [10], [11], [12]. Hence, inhibiting EMT by targeting the signaling pathways involved is a potential therapeutic strategy for cancer treatment [13], [14], [15]. Hyperactivation of Wnt signaling is known to contribute to EMT in human cancer [16], [17], [18], [19], [20]. The Wnt pathway is regulated by Hippo signaling [21]. Non-phosphorylated (active) forms of YAP/TAZ along with (TEAD) act coordinately or with Smad proteins or β-catenin/T cell factor to induce genes that regulate cell migration and cancer metastasis [22], [23], [24]. Upstream kinases Lats phosphorylate (inactivate) Hippo effector proteins, resulting in their cytoplasmic retention followed by proteasomal degradation by the glycogen synthase kinase (GSK3)-β complex or in sequestration of β-catenin and Smads, which blocks the activation of these transcription factors [20].
Micro (mi)RNAs regulate a variety of physiological processes as well as cancer development [25], [26]. MiRNA dysregulation has been implicated in OS progression [27]. We previously reported that YAP directly induces miR-130a, resulting in increased YAP activity [28]. In the present study, we investigated miRNA-mediated regulation of Hippo pathway components and found that miR-135b expression is induced by TAZ and in turn inhibits LATS2, adenomatous polyposis coli (APC), and GSK-3β protein expression. A TAZ-miR-135b positive feedback loop amplified upstream signals controlling cell proliferation and EMT. TAZ inactivation by miR-135b suppressed tumorigenesis. These results suggest that miR-135b inhibition is a novel therapeutic strategy for treatment of OS.
Section snippets
Ethics
All animal experiments were carried out according to relevant national and international guidelines and approved by the Stanford Institutional Animal Care and Use Committee (IACUC). All experiments strictly followed the panel's specific guidelines regarding the care, treatment and euthanasia of animals used in the study.
Clinical samples
Slices of formalin-fixed and paraffin-embedded primary osteosarcoma and chondroma tissues were obtained from biopsies in 16 and 20 patients before administration of neo-adjuvant
TAZ overexpression is associated with cell migration and a mesenchymal phenotype
We investigated the role of TAZ in OS cell migration with the Matrigel assay using the U2OS and HOS cell lines, which have high migratory capacity (Fig. 1A and B). We found that TAZ mRNA and protein levels were increased in U2OS and HOS cell sublines (Fig. 1C and D). Previous studies have demonstrated that EMT and cell migration are associated with tumor metastasis; we therefore examined TAZ and EMT marker expression in OS cells. We found that TAZ expression was negatively correlated with that
Discussion
EMT generates an aggressive tumor phenotype that leads to local metastasis via direct invasion or to distant metastasis via lymphatic and circulatory systems [31]. Tumor metastasis can presumably be inhibited by targeting factors involved in EMT [15], [32], [33]. The present study showed that EMT and tumorigenesis in OS are associated with TAZ upregulation. Moreover, we identified a novel TAZ–miR-135b positive feedback mechanism (Fig. 7F) that maintains EMT via amplification of Hippo signaling
Acknowledgements
The study was sponsored by the National Nature Science Fund of China (81601925, 81472064 and 61502129), the Zhejiang Provincial Natural Science Foundation of China (No. LQ16F020004), Medical Science and Technology Project of Zhejiang Province (2017179447). No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this study.
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