CELL‐MATRIX INTERACTIONS AND MECHANOSENSITIVE PATHWAYS IN UTERINE LEIOMYOMA (FIBROID) PROLIFERATION

Abstract

BackgroundUterine leiomyomas (fibroids; UF) are benign myometrial neoplasms that produce clinically‐relevant symptoms in >25% of all women including excessive menstrual bleeding and infertility, and represent the leading cause of hysterectomy (~600,000/year in the US). Despite their importance, the mechanisms that promote UF growth are still not well‐understood. UFs are comprised of smooth muscle cells and fibroblasts with substantial extracellular matrix (ECM). While cells within UFs produce ECM, the role of ECM per se in modulating cell properties towards overall tumor growth is not known. This study tests the hypothesis that UF cells are mechanically sensitive, and that the ECM provides a positive feedback mechanism which further promotes UF growth.MethodsCells were enzymatically isolated from UFs of patients undergoing myomectomy at Mayo Clinic Rochester (IRB approved). Fibroid cells were plated onto soft (0.2kPa) vs. stiff (64kPa) polydimethylsiloxane (PDMS) elastomer substrates or standard tissue culture plastic substrates (~100GPa) for 24 h in DMEM containing 10% FBS prior to RNA isolation and qPCR analysis. Transcript levels for known ECM and profibrotic genes were determined. Nuclear localization of YAP and TAZ was visualized using immunofluorescence and quantified using a Cytation5 imaging system. Separately, 2‐color fluorescence immunohistochemistry for YAP and TAZ was performed on formalin fixed; paraffin embedded normal myometrium and fibroid tissue sections. Transciption level and nuclear localization calculations were performed with Kruskall‐Wallis and Wilcoxon Sign‐Rank tests, respectively.ResultsSignificant increases in transcripts for ECM gene Collagen 1 (COL1A1) were noted for UF cells plated on stiff PDMS and standard plastic compared to soft PDMS. The pro‐fibrotic genes Connective Tissue Growth Factor (CTGF), alpha smooth muscle actin (ACTA2), Endothelin 1 (EDN1), and plasminogen activator inhibitor 1 (SERPINE1) were progressively increased in expression going from soft PDMS substrate to plastic. The anti‐fibrotic gene, cyclooxygenase 2 (PTGS2) was decreased on stiff PDMS and plastic compared to soft PDMS. In cells plated on plastic, there was a significant decrease in YAP/TAZ nuclear localization in cells treated with the Rho‐kinase inhibitor Y‐27632, consistent with a mechanobiologic feedback. Immunohistochemistry showed a two‐fold increase in YAP/TAZ nuclear localization for fibroid tissue compared to normal myometrium.ConclusionsBased on the growing appreciation that ECM mechanical properties contribute to UF pathobiology and fibrotic matrix deposition, we show enhanced ECM and profibrotic gene expression by fibroid cells as a function of substrate stiffness. These results demonstrate a feedback mechanism by which UFs progress through matrix stiffness‐mediated ECM deposition. Here, the mechanosensitive pathways of YAP and TAZ may be involved in transducing ECM signals to UF growth.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.