Modulation of AKT activity is associated with reversible dormancy in ascites-derived epithelial ovarian cancer spheroids

Abstract

Epithelial ovarian cancer (EOC) metastasis is a direct contributor to high recurrence and low survival for patients with this disease. Metastasis in EOC occurs by cell exfoliation from the primary tumor into the fluid-filled peritoneal cavity, persistence of these cells as non-adherent multicellular aggregates or spheroids and reattachment of spheroids to form secondary lesions. We have recovered native spheroids from ascites fluid and demonstrated that EOC cells within these structures exhibit reduced proliferation, yet regain the capacity to attach and reinitiate cell division. To model this process in vitro for further investigation, primary EOC cells from patient peritoneal fluid were cultured under non-adherent conditions. Here we show that these cells naturally form spheroids resembling those observed in ascites. Spheroids exhibit reduced cell proliferation and a protein expression pattern consistent with cellular quiescence: specifically, decreased phospho-AKT and p45/SKP2 with a concomitant increase in p130/RBL2 and p27(Kip1). However, when spheroids are seeded to an adherent surface, reattachment occurs rapidly and is followed by reinitiation of AKT-dependent cell proliferation. These results were strikingly consistent among numerous clinical specimens and were corroborated in the EOC cell line OVCAR3. Therefore, our data reveal that EOC cells become quiescent when forming spheroids, but reactivate proliferative mechanisms upon attachment to a permissive substratum. Overall, this work utilizes a novel in vitro model of EOC metastasis that employs primary human EOC cells and introduces the important concept of reversible dormancy in EOC pathogenesis.

BMP signalling controls the malignant potential of ascites-derived human epithelial ovarian cancer spheroids via AKT kinase activation

“…Our laboratory has independent evidence that the PI3 K-AKT pathway is down-regulated endogenously during EOC spheroid formation, yet its activation is required again during spheroid reattachment and cell dispersion [28]. Taken together, AKT and BMP signalling are co-ordinately down-regulated during EOC spheroid formation.…”

“…5c). We and others have demonstrated that active PI3 KmTOR signalling is vital to promoting the metastatic potential of EOC cells and spheroids [26][27][28][29]. Indeed, treatment of reattaching primary EOC spheroids with any of three different inhibitors of the PI3 K-mTOR pathway, LY294002, rapamycin and temsirolimus (Torisel Ò ), resulted in a significant reduction in cell dispersion (Suppl.…”

Mechanisms of disseminated cancer cell dormancy: an awakening field

“…Floating tumour spheroids of ovarian cancer cell lines or spheroids isolated directly from patients’ peritoneal fluid (ascites) were shown by one study to enter a G0/G1 arrest 23 . This arrest was due to AKT inhibition and increased p130 (also known as RBL2) and p27 (also known as KIP1) protein levels, which sequester RB and inhibit cyclin-dependent kinase 4 (CDK4) and/or CDK6 activity to induce G0/G1 arrest.…”

Dormant Cells: The Original Cause of Tumor Recurrence and Metastasis

“…Quiescent D2.0R cells are induced to proliferate through b1-integrin activation of SRC and focal adhesion kinase (FAK), leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Dual signaling by fibroblast growth factor (FGF-2) which acts directly through phosphatidylinositol 3-kinase (PI3 K) and integrin a5b1 is also necessary for cortical rearrangement in dormant cells [20].…”