title: An mDia2/ROCK signaling axis regulates invasive egress from epithelial ovarian cancer spheroids.
abstract: Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, DRF3, has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.
authors.affiliation: Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, Ohio, United States of America.
authors.forename: Krista M
authors.initials: KM
authors.lastname: Pettee
authors.affiliation: Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, Ohio, United States of America.
authors.forename: Kaitlyn M
authors.initials: KM
authors.lastname: Dvorak
authors.affiliation: Department of Surgery, University of Toledo Health Science Campus, Toledo, Ohio, United States of America.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Nestor-Kalinoski
authors.affiliation: Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, Ohio, United States of America.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Eisenmann
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000077216:Carcinoma, Ovarian Epithelial; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D005260:Female; D000080865:Formins; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009375:Neoplasms, Glandular and Epithelial / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018874:Spheroids, Cellular / Q000378:metabolism* / Q000473:pathology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C491462:DIAPH2 protein, human; D000080865:Formins; D034741:RNA, Small Interfering; C066737:RHOA protein, human; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein