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.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5′-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence.

Epithelial ovarian cancer (EOC) cells have the ability to form multi-cellular aggregates in malignant ascites which dramatically alters cell signalling, survival, and metastatic potential. Herein, we demonstrate that patient ascites-derived EOC cells down-regulate endogenous bone morphogenetic protein (BMP) signalling by decreasing BMP ligand expression when grown in suspension culture to form spheroids. Enforced BMP signalling in these cells via constitutively-active BMP type I ALK3(QD) receptor expression causes the formation of smaller, more loosely-aggregated spheroids. Additionally, ALK3(QD)-expressing spheroids have an increased rate of adhesion and dispersion upon reattachment to substratum. Inhibition of endogenous BMP signalling using recombinant Noggin or small molecule inhibitor LDN-193189, on the other hand, opposed these phenotypic changes. To identify potential targets that impact the phenotype of EOC spheroids due to activated BMP signalling, we performed genome-wide expression analyses using Affymetrix arrays. Using the online Connectivity Map resource, the BMP signalling gene expression signature revealed that the AKT pathway is induced by activated BMP signalling in EOC cells; this finding was further validated by phospho-AKT immuno-blotting. In fact, treatment of EOC spheroids with an AKT inhibitor, Akti-1/2, reduced BMP-stimulated cell dispersion during reattachment as compared to controls. Thus, we have identified AKT as being one important downstream component of activated BMP signalling on EOC spheroid pathobiology, which may have important implications on the metastatic potential of this malignancy.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2 versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p=0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

Purpose. In order to meet a clinical need for better pathways to access genetic testing for ovarian cancer patients, we implemented and reviewed an opt-out referral process for genetic consultation whereby a referral is automatically sent to genetics following a pathological diagnosis of HGSC. Methods. Following implementation of the opt-out referral process, each month a list of new cases of HGSC was generated from the synoptic pathology report and forwarded directly to the Cancer Genetics clinic. Using an advanced directive, patients were automatically referred for genetic counselling two months after surgery. If the patient declined genetic counselling (opted-out) after discussion with their surgeon within the two months after surgery, the Genetic Counsellor was informed and the patient was removed from the referral process. Results. Between January 1, 2015, and December 31, 2017, 168 women were diagnosed with HGSC, of whom 167 received a referral for genetic consultation. In only one case the referral was cancelled by the surgeon, resulting in a referral rate of 99.4%. By the end of the study period, 133 women attended a genetics consultation appointment and 125 (94%) agreed to proceed with genetic testing. Among those who completed genetic testing, 15% tested positive for a BRCA1 or BRCA2 gene mutation. Of the women who tested positive for a BRCA1/2 mutation, 56% had no family history of breast or ovarian cancer. Conclusions. The opt-out referral process described in this study is s a feasible, effective, and patient-centred approach to increase access to BRCA1/2 testing for patients with ovarian cancer.

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