LAUSR

BACKGROUND Uterine leiomyomas, the most common tumors of the female reproductive system, feature excessive deposition of disordered stiff extracellular matrix (ECM) and fundamentally altered mechanical signaling pathways . Specifically, these alterations impact the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted via integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress β1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an anti-hyperlipidemic drug, has anti-leiomyoma effects through cellular, animal model and epidemiologic studies. OBJECTIVE The purpose of this study was to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from five patients who underwent hysterectomy at the Department of Gynecology and Obstetrics at the Johns Hopkins Hospital. Primary and immortalized human leiomyoma (HuLM) cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, 1μM or control) for 48 h. Protein and mRNA levels of β1 integrin and ECM components involved in mechanical signaling were quantified by qRT-PCR, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on RhoA activity using pull-down assay and gel contraction. RESULTS We found that simvastatin significantly reduced the protein expression of β1 integrin by 44% and type I collagen by 60% compared to untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of FAK down to 26-60% of control whereas it increased total FAK protein expression. Using a RhoA pull-down activation assay, we observed that simvastatin-treated cells had reduced levels of active RhoA by 45-85% compared to control. Consistent with impaired RhoA activation, simvastatin treatment reduced tumor gel contraction where gel area was 122-153% larger compared to control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in β1 integrin downstream signaling, including AKAP13, ROCK1, MLCK, and cyclin D1. CONCLUSION The results of this study suggest a possible therapeutic role for simvastatin to restore the altered state of mechanotransduction signaling in leiomyoma. Collectively, the findings are aligned with previous epidemiologic and other reports and support the need for clinical trials.