Multicellular aggregates of malignant cells, or spheroids, are frequently present in ascites of patients with advanced-stage EOC. These structures facilitate metastatic transit and promote secondary tumour establishment, and many molecular features of spheroid cells define their dormant, chemo-resistant phenotype. As such, a more complete understanding of EOC disease progression requires knowledge of the molecular changes that occur during metastasis, from detachment and spheroid formation to establishment of secondary tumours. We have discovered many altered biological pathways in our spheroid model of EOC metastasis, and herein we describe how MYC oncoprotein abundance and its transcriptional activity are rapidly and reversibly downregulated in EOC spheroids to contribute to their dormant state.
While MYC has been extensively studied in the context of cancer, little is known about its regulation within the three-dimensional context of EOC spheroids. However, several stress response pathways that we have characterized in the context of EOC spheroids have been shown to impact MYC protein abundance in other studies. AMP-activated protein kinase (AMPK) is a central mediator of energy homeostasis38 and is canonically activated by Liver Kinase B1 (LKB1)39 in response to decreased ATP levels40,41. We have demonstrated previously that EOC spheroids exhibit decreased ATP with concomitant elevated AMPK activation42, that occurs rapidly after cell detachment in a LKB1-independent and CAMKK2-dependent manner43,44. In addition, metformin, an indirect activator of AMPK, leads to increased phosphorylation of MYC at Thr58 to decrease MYC abundance in prostate cancer cells45. Thus, MYC depletion in EOC spheroids may occur due to AMPK activation upon cell detachment.
Oxygen gradients have been reported in the context of multicellular spheroids, with cells situated in the spheroid interior often exposed to diminished oxygen levels compared to cells at the periphery. Particularly in larger spheroids, this feature may be accompanied by the formation of a hypoxic core46. Low oxygen levels promote stabilization of HIF-147–49, leading to broad changes in gene expression that enable adaptation to hypoxic conditions50. Independent of HIF-1-mediated transcriptional responses, hypoxia has been shown to induce phosphorylation of MYC at Thr58 and resultant proteasome-mediated MYC depletion in osteosarcoma (U2OS), cervical cancer (HeLa), and lung cancer (H460) cells51. Consequently, intracellular stress responses to energy depletion and hypoxia may contribute to sustained MYC suppression in EOC spheroids.
Nevertheless, while such stressors associated with multicellular spheroids can affect MYC levels, they are unlikely to be the primary immediate triggers in our model, given the rapid depletion observed in our time course studies. As such, our findings underscore a functional relationship between cell attachment and proteasomal degradation of MYC in EOC cells, and implicate detachment as the main immediate trigger for MYC degradation in EOC spheroids. While our study has not determined the precise signaling mechanisms linking cell attachment to MYC abundance in EOC, plausible factors include signaling mediators downstream from cell adhesion molecules (CAMs) and their extracellular matrix (ECM) substrates as these would be immediately perturbed upon cell detachment. For example, glycogen synthase kinase 3-beta (GSK3-beta)52, a kinase known to phosphorylate MYC at Thr5853, is negatively regulated by integrin-linked kinase (ILK)52,5554, suggesting a possible mechanism whereby loss of cell-matrix attachment would promote degradation of MYC in EOC cells. In a reciprocal fashion, breast epithelial cells utilize beta-1-integrin-dependent cell adhesion to the ECM proteins fibronectin and collagens type I and IV to induce MYC protein levels56. Further mechanistic investigation of cell adhesion and MYC protein regulation in EOC is warranted, since it may improve our understanding of how the seeding of spheroids at metastatic sites trigger their renewed growth potential.
As mentioned above, downregulation of MYC protein and its transcriptional activity that we observed in EOC spheroids parallels other molecular mechanisms aligned with tumour dormancy. EOC spheroid cells exhibit a reversible dormancy phenotype characterized by reduced proliferation, increased resistance to anoikis and chemotherapeutic agents, and several molecular changes that control cellular quiescence. These include decreased p-AKT (S473) and p54/SKP2 along with increased p130/RBL2 and p27Kip1, all of which are reversed upon reattachment22. Given MYC’s central role in promoting cell proliferation, its suppression would be a plausible strategy for EOC spheroids to adopt during acquisition of dormancy. MYC depletion has been shown to inhibit proliferation in numerous human tumour cell lines57, and our data show that MYC knockdown increases doubling time in COV362 and ES-2 cells. Important to our working model of reversible dormancy, MYC depletion occurs rapidly upon cell detachment but its expression and transcriptional activity are restored by reattachment, strengthening the idea that these properties are connected. As such, suppression of MYC activity may have numerous consequences controlling dormancy in EOC spheroids.
Notably, while our results indicate decreased activity in spheroids for the majority of EOC cell lines, a small subset of lines exhibit little or no change. We noted a similar trend in our analysis of published HGSOC scRNA-Seq data, where MYC-associated transcriptional signature scores were substantially decreased in ascites-derived as compared with matched primary tumour-derived EOC cells in one patient, but this effect was less so for the other. Given the central role of MYC in promoting cell proliferation and metabolism, a lack of MYC downregulation in some EOC patient ascites cells could serve as a biomarker indicating their reduced spheroid dormancy status and thus have retained sensitivity to cytotoxic chemotherapies.
Our findings suggest a scenario where MYC activity promotes EOC cell proliferation within primary tumours, but this is transiently suppressed upon detachment during metastatic transit, and is restored upon reattachment and establishment of metastases. We propose that reversible, detachment-induced downregulation of MYC is a key feature of EOC spheroid dormancy contributing to their quiescent-like phenotype. If MYC downregulation is an essential response to loss of EOC cell anchorage, elucidating the molecular mechanisms that link cell detachment to MYC downregulation may reveal novel ways of impacting both metastatic potential and chemotherapy resistance.