Role of MicroRNA-29 and ADAM12 in the Regulation of REST Dependent Signaling Pathways in Uterine Fibroids

Abstract

Uterine fibroids, also known as leiomyomas, are benign smooth muscle cell (SMC) tumors of the myometrium and are the most frequent reason for a hysterectomy. Although benign, these tumors pose a significant burden on patients with symptoms of abdominal pain, pressure, uterine bleeding, and infertility; creating a great liability for the US economy with an annual estimated cost of up to $34 billion. Currently there are no long-term treatments for fibroids that will leave fertility intact, mainly because the mechanism of pathogenesis of these tumors is largely unknown. One of the key characteristics of uterine fibroids is the excessive deposition and reorganization of extracellular matrix (ECM). Altered ECM, which amplifies growth factor signaling and disrupts mechanosensing, has been proposed to promote fibroid tumor growth. Analysis of available gene expression datasets from GEO database indicates that ADAM12 expression is dramatically upregulated in fibroids. As a member of the A-Disintegrin And Metalloprotease family of matrix modifying enzymes, ADAM12 is known to cleave ECM proteins, activate epidermal growth factor (EGF) and Insulin-like growth factor (IGF) signaling and promote tumorigenesis. Our lab has recently shown the expression of RE1 suppressing transcription factor /neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, to be lost in fibroids. Upon further analysis, we found silencing REST in primary myometrial SMCs led to an increase in a number of downstream target genes, a profile similar to what is seen in fibroid tumor samples. Furthermore, Ingenuity® pathway analyses of gene expression datasets for fibroids indicate that the loss of REST and the increase in ADAM12 expression in leiomyomas could be linked through microRNA-29 (miR-29). In addition, in cultured mammary tumor cell lines, miR-29 is known to directly regulate ADAM12 expression. Compared to normal myometrium, uterine fibroids express significantly lower levels of miR-29. In its promoter, miR-29 contains an RE1 element, a putative binding site for REST. In order to see how the loss of REST in fibroids could affect miR-29, we silenced REST in primary myometrial SMCs and saw a significant decrease in miR-29 expression. In addition, Western blot analysis showed an increase in ADAM12 expression and EGF receptor (EGFR) phosphorylation when REST was knocked-down in primary myometrial cells. We further investigated the link between miR-29 and ADAM12 expression by treating primary myometrial and leiomyoma cells with miR-29 inhibitors and mimics, respectively. We found that inhibiting miR-29 in myometrial cells results in an increase in the expression of ADAM12. Conversely, the opposite effects were seen when fibroid cells were treated with miR-29 mimics. Furthermore, overexpression of ADAM12 in primary myometrial SMCs was found to induce activation of multiple tumorigenic signaling pathways including EGFR, Mitogen-activated protein kinase (MAPK), the phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/Akt), and mammalian target of rapamycin (mTOR) and Notch pathways. In conclusion, we report a novel druggable pathway that links the loss of REST, down regulation of miR-29, increased ADAM12 expression, and the activation of multiple tumorigenic pathways in uterine fibroids.