Norepinephrine promotes anoikis resistance in fallopian tube secretory cells through beta adrenergic receptor signaling (247)

Abstract

<b>Objectives:</b> High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy with a 5-year survival rate approaching 40%. Unfortunately, most women present at an advanced stage due to subtle symptomatology at disease onset and inadequate screening protocols. Thus, understanding the cellular events that lead to the development and malignant transformation of fallopian tube (FT) precursor cells into metastatic disease may lead to actionable targets to improve outcomes. Fallopian tube secretory epithelium is considered the cell of origin of HGSOC, where <i>TP53</i> and other genetic defects accrue to permit exfoliation and seeding of the ovary and peritoneum, leading to widespread metastasis. Follicular fluid bathes the fallopian tube monthly during the reproductive years and contains several factors contributing to carcinogenesis. Recently, neurotransmitters such as norepinephrine (NE) have been implicated in ovarian cancer growth and metastasis through multiple mechanisms and are also present within the follicular fluid. Thus, the objective of the current study was to evaluate the effects of NE on FT epithelial cell function under 3D conditions. <b>Methods:</b> FT cell lines (FT189, FT190, FT194, FT237, FT240, FT246, FT282), previously immortalized through several mechanisms, were plated into round bottom ultralow adhesion plates with 10µM NE or vehicle control (VHC), in the presence or absence of adrenergic antagonist propranolol (10µM). Spheroids were serially imaged over 24h. For FT237 and FT246, live/dead cell staining was performed using ReadyProbe cell viability assay, and apoptotic activity was assessed via annexin staining analyzed by flow cytometry. qPCR and western blotting were used to assess receptor expression. <b>Results:</b> All FT lines examined expressed <i>ADRB1</i> and <i>ADRB2</i> by both western blot and qPCR. Treatment with NE resulted in significant compaction of spheroid formation by 33% (+/- 5%, p <0.001) at 24h compared to VHC. This response was completely abrogated by preexposure to propranolol (p <0.05). NE nor propranolol had a significant impact on cell viability under immunofluorescence. However, annexin staining revealed a significant 5% increase in cell survival with a concomitant decrease in early and late apoptotic cell populations exposed to NE in 3D culture (p<0.05), compared to VHC. FT exposure to NE resulted in significantly increased expression of EMT genes, <i>SNAI1</i> and <i>SNAI2</i>, which was completely reversed by pretreatment with propranolol (p<0.05). <b>Conclusions:</b> Taken together, these findings demonstrate that NE exposure promotes FT cell spheroid formation and EMT gene transcription through activation of ADRB-mediated signaling. Thus, NE may potentiate metastasis of early ovarian cancer precursor lesions through stimulation of EMT genes and enhancing anoikis resistance, two factors that are critical to the coelomic spread of ovarian cancer. Future studies examining NE-induced carcinogenesis in FT cells are warranted.