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.

mDia2 formin selectively interacts with catenins and not E-cadherin to regulate Adherens Junction formation

“…3B), indicating a formin-specificity to this interaction. This is consistent with previous findings suggesting that mDia2, and not mDia1, is involved in junctional stability in ovarian cancer spheroids [44]. Neither mDia1 nor mDia2 precipitated with the cadherins (Fig.…”

“…Initially at 30 minutes and 1 hour, control cells formed larger and more loosely-packed spheroids compared to mDia2 KD cells. This is consistent with previous findings indicating that mDia2 depletion in ovarian cancer cells led to increased spheroid compaction [44]. However, by 2 hours, mDia2 KD and control cells formed similar sized spheroids (Fig.…”

“…The poor prognosis associated with EOC can largely be attributed to its diagnosis in the late stages of the disease when cancer cells have already disseminated within the peritoneal cavity. Previously, depletion of mDia2, but not mDia1, was associated with single cell invasion from ovarian cancer spheroids [44]. Here, we show for the first time that mDia2 is essential for maintenance of cell-cell junction strength in EOC spheroids and AJ formation.…”

“…In ovarian cancer, decreased E-cadherin expression is associated with invasive peritoneal seeding and is more commonly observed in borderline ovarian tumors and carcinomas compared to benign tumors [43]. Previously, mDia2 depletion promoted ovarian cancer spheroid invasion by driving single cell invasive egress from the spheroid [44]. The exact mechanisms of action remained elusive, however.…”

mDia2 and CXCL12/CXCR4 chemokine signaling intersect to drive tumor cell amoeboid morphological transitions

“…Rho GTPase activation regulates amoeboid blebbing, and is mediated by various extracellular signals [23]. RhoA promotes amoeboid transitions in tumor cells through RhoA, ROCK and myosin light chain [24–27]. Rho GTPases are regulated via GEFs and GTPase-activating proteins (GAPs), which modulate GTPase activity.…”