NAV3, a Tumor Suppressor Gene, Is Decreased in Uterine Leiomyoma Tissue and Cells

Abstract

NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.

Keywords: Cetrorelix; GnRH; Leiomyoma; Leuprolide acetate; Myometrium; NAV3; Tumor suppressor gene.

Fig. 1

NAV3 mRNA and protein expression in untreated cell line and surgical samples. Relative expression of NAV3 mRNA was decreased in leiomyoma cells as compared against myometrial cells from patient tissue (n = 14 patient-matched leiomyoma and myometrium pairs) (A). The same was found in tissue samples from cell line leiomyoma as compared against myometrium (B). Results are presented as fold change with non-targeting controls (NTC) as the reference, mean ± SEM of three independent experiments. *Statistically significant difference, p < 0.05. In the majority of patients, NAV3 protein was decreased in leiomyomas with the exception of one patient, in which the result found similar protein expression in both leiomyoma and myometrial samples. NAV3 Western data are presented for each individual patient to best illustrate this heterogeneity. Data shown represent mean ± standard error of mean (SEM) fold. Results were normalized to CoxIV (p = 0.0009) (C)

Fig. 2

NAV3 protein expression by immunohistochemistry in leiomyoma and adjacent myometrial surgical specimens. Representative sample of leiomyoma surgical specimen and the adjacent myometrium in four representative patient pairs: patient 1 myometrium (A), patient 1 leiomyoma (B); patient 2 myometrium (C), patient 2 leiomyoma (D); patient 3 myometrium (E), patient 3 leiomyoma (F); patient 4 myometrium (G), patient 4 leiomyoma (H). Images were obtained at × 20 magnification, demonstrating more prominent NAV3 staining (brown staining) in myometrial specimens as compared with leiomyoma specimens. Specimens (n = 14 patient-matched leiomyoma and myometrial pairs) were quantitated using the H-score method, and demonstrated a quantitative decrease in leiomyoma compared with myometrium (H-score 154.7 ± 6.2 vs 312.5 ± 14.7, P < 0.0001)(I)

Fig. 3

NAV3 mRNA and protein expression in leuprolide acetate– and cetrorelix-treated leiomyoma cells. NAV3 mRNA expression was found to be increased in treated leiomyoma compared against untreated leiomyoma with 5 days of LA treatment (A). Western blot analysis of NAV3 expression in treated leiomyoma cells (× 2 biologic replicates). Five-day treatment with LA increases NAV3 expression in leiomyoma (B). 72-h treatment with cetrorelix increases NAV3 protein expression in leiomyoma at 10⁻¹⁰ M concentration (C). Results were normalized to CoxIV. Data shown represent mean fold ± SD

Fig. 4

Proposed GnRH-analog effect on NAV3 and resultant effect on leiomyoma development. It has prior been established that in a typical leiomyoma cell, GnRHR related pathway signaling is upregulated. This results in activated b-catenin contributing to tumor growth. Additionally, activated GnRHR pathways in the leiomyoma include upregulation of T cell factors which increase inflammation and also contribute to fibroid volume. With GnRH analog treatment GnRH receptors become downregulated, resulting in decreased GnRHR-mediated processes. We discovered that GnRH analogs increase NAV3 expression in fibroids. Based on NAV3 functioning in other tissues, the increase in NAV3 may affect the GnRHR pathway signaling through the mechanism of a feedback loop, ultimately resulting in decreased GnRHR-mediated tumor growth. It is currently unknown if NAV3 is a byproduct of GnRHR pathway signaling or if it is a mediator. Currently, the most commonly accepted mechanism of action of GnRH analogs on leiomyoma is through the pituitary-mediated decrease in estrogen and progesterone. This represents the canonical or known pathway. Our results highlight the potential hormone-independent mechanisms, for how GnRH analogs decrease fibroid volume, by downregulated GnRHR pathways which may be mediated by an increase in NAV3, (non-canonical pathway)