Gabriel E. DiMattia

Transcriptomic Analyses of Ovarian Clear Cell Carcinoma Spheroids Reveal Distinct Proliferative Phenotypes and Therapeutic Vulnerabilities

Cancer cell spheroids autonomously form in the ascites fluid and are considered a conduit for epithelial ovarian cancer metastasis within the peritoneal cavity. Spheroids are homotypic, avascular 3D structures that acquire resistance to anoikis to remain viable after cellular detachment. We used in vitro spheroid model systems to interrogate pathways critical for spheroid cell proliferation, distinct from those driving monolayer cancer cell proliferation. Using the 105C and KOC-7c human ovarian clear cell carcinoma (OCCC) cell lines, which have distinct proliferative phenotypes as spheroids but the same prototypical OCCC gene mutation profile of constitutively activated AKT signaling with the loss of ARID1A, we revealed therapeutic targets that efficiently kill cells in spheroids. RNA-seq analyses compared the transcriptome of 3-day monolayer and spheroid cells from these lines and identified the characteristics of dormant spheroid cell survival, which included the G2/M checkpoint, autophagy, and other stress pathways induced in 105C spheroids, in sharp contrast to the proliferating spheroid cells of the KOC-7c cell line. Next, we assessed levels of various G2/M checkpoint regulators and found a consistent reduction in steady-state levels of checkpoint regulators in dormant spheroid cells, but not proliferative spheroids. Our studies showed that proliferative spheroid cells were sensitive to Wee1 inhibition by AZD1775, but the dormant spheroid cells showed a degree of resistance to AZD1775, both in terms of EC50 values and spheroid reattachment abilities. Thus, we identified biomarkers of dormant spheroids, including the G2/M checkpoint regulators Wee1, Cdc25c, and PLK1, and showed that, when compared to proliferating spheroid cells, the transcriptome of dormant OCCC spheroids is a source of therapeutic targets.

Exploiting Cancer Dormancy Signaling Mechanisms in Epithelial Ovarian Cancer Through Spheroid and Organoid Analysis

Epithelial ovarian cancer (EOC) exhibits a unique mode of metastasis, involving spheroid formation in the peritoneum. Our research on EOC spheroid cell biology has provided valuable insights into the signaling plasticity associated with metastasis. We speculate that EOC cells modify their biology between tumour and spheroid states during cancer dormancy, although the specific mechanisms underlying this transition remain unknown. Here, we present novel findings from direct comparisons between cultured EOC spheroids and organoids. Our results indicated that AMP-activated protein kinase (AMPK) activity was significantly upregulated and protein kinase B (Akt) was downregulated in EOC spheroids compared to organoids, suggesting a clear differential phenotype. Through RNA sequencing analysis, we further supported these phenotypic differences and highlighted the significance of cell cycle regulation in organoids. By inhibiting the G2/M checkpoint via kinase inhibitors, we confirmed that this pathway is essential for organoids. Interestingly, our results suggest that specifically targeting aurora kinase A (AURKA) may represent a promising therapeutic strategy since our cells were equally sensitive to Alisertib treatment as both spheroids and organoids. Our findings emphasize the importance of studying cellular adaptations of EOC cells, as there may be different therapeutic targets depending on the step of EOC disease progression.

NOTCH Signaling Limits the Response of Low-Grade Serous Ovarian Cancers to MEK Inhibition

Abstract Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian cancer with high fatality rates in advanced stages due to its chemoresistant properties. LGSOC is characterized by activation of MAPK signaling, and recent clinical trials indicate that the MEK inhibitor (MEKi) trametinib may be a good treatment option for a subset of patients. Understanding MEKi-resistance mechanisms and subsequent identification of rational drug combinations to suppress resistance may greatly improve LGSOC treatment strategies. Both gain-of-function and loss-of-function CRISPR-Cas9 genome-wide libraries were used to screen LGSOC cell lines to identify genes that modulate the response to MEKi. Overexpression of MAML2 and loss of MAP3K1 were identified, both leading to overexpression of the NOTCH target HES1, which has a causal role in this process as its knockdown reversed MEKi resistance. Interestingly, increased HES1 expression was also observed in selected spontaneous trametinib-resistant clones, next to activating MAP2K1 (MEK1) mutations. Subsequent trametinib synthetic lethality screens identified SHOC2 downregulation as being synthetic lethal with MEKis. Targeting SHOC2 with pan-RAF inhibitors (pan-RAFis) in combination with MEKi was effective in parental LGSOC cell lines, in MEKi-resistant derivatives, in primary ascites cultures from patients with LGSOC, and in LGSOC (cell line–derived and patient-derived) xenograft mouse models. We found that the combination of pan-RAFi with MEKi downregulated HES1 levels in trametinib-resistant cells, providing an explanation for the synergy that was observed. Combining MEKis with pan-RAFis may provide a promising treatment strategy for patients with LGSOC, which warrants further clinical validation.