Distinct roles of BMP and LKB1/AMPK signalling impacting ovarian cancer spheroid biology

High-grade serous (HGS) carcinoma, the most prevalent and most deadly subtype of epithelial ovarian cancer (EOC), presents unique therapeutic challenges since the majority of cases are diagnosed at advanced, metastatic stage. At this point widespread intraperitoneal metastatic lesions are numerous, which is why models that recapitulate disease dissemination are critical to uncover novel therapeutic targets. One of the initiating events in ovarian cancer metastasis is shedding from the primary tumour into the peritoneal cavity where cells must survive in suspension in order to seed secondary tumours. This non-adherent population of cells exists as multicellular aggregates, or spheroids; data from our lab has demonstrated that cells within spheroids are dormant, yet are readily alter their phenotype upon reattachment to an adherent substratum. To further explore the pathobiology of ovarian cancer spheroids, my thesis work describes the functional characterization of two different signalling pathways— bone morphogenetic protein (BMP), and the liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK)—which mediate distinct and important aspects of spheroid formation and reattachment. Activated BMP signalling resulted in smaller, loosely-aggregated spheroids, which were more readily able to reattach and disperse. These phenotypic alterations observed as a result of active BMP signalling were mediated, at least in part, by cooperation with the AKT signalling pathway. These studies implicate inhibition of BMP and AKT signalling as potential strategies for therapeutic targeting of reattaching spheroids, which is critical for the formation of secondary metastatic lesions. Other work in our lab implicated the downregulation of AKT signalling in spheroid formation-induced dormancy. In an attempt to uncover additional pathways promoting the dormant phenotype of ovarian cancer spheroids, I investigated the LKB1/AMPK signalling cascade given its ability to alter cellular metabolism in response to nutrient and energy availability. Despite a dramatic enhancement in AMPK activity observed in ovarian cancer spheroids, targeted knockdown had no effect on viability of cells in this context. However, knockdown of its upstream kinase, LKB1, revealed a dramatic decrease in ovarian cancer spheroid viability, suggesting a role for this kinase in mediating anoikis-resistance in an AMPK-independent manner. Taken together, my results have uncovered two distinct and important signalling pathways that regulate unique aspects of spheroid formation, cell survival, and reattachment. By understanding the molecular mechanisms used by ovarian cancer spheroids to survive during dissemination and promote secondary metastasis, my work has uncovered additional therapeutic targets for the potential treatment of advanced-stage ovarian cancer

Direct Genetics Referral Pathway for High-Grade Serous Ovarian Cancer Patients: The opt-Out Process

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

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids

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

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability

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. © The Author 2014. Published by Oxford University Press. All rights reserved

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Direct Genetics Referral Pathway for High-Grade Serous Ovarian Cancer Patients: The “Opt-Out” Process

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

Effects of Age, Gender, BMI, and Anatomical Site on Skin Thickness in Children and Adults with Diabetes

<div><p>Objective</p><p>We aimed to assess the effects of age, sex, body mass index (BMI), and anatomical site on skin thickness in children and adults with diabetes.</p><p>Methods</p><p>We studied 103 otherwise healthy children and adolescents with type 1 diabetes aged 5–19 years, and 140 adults with type 1 and type 2 diabetes aged 20–85 years. The thicknesses of both the dermis and subcutis were assessed using ultrasound with a linear array transducer, on abdominal and thigh skin.</p><p>Results</p><p>There was an age-related thickening of both dermis (p&lt;0.0001) and subcutis (p = 0.013) in children and adolescents. Girls displayed a substantial pubertal increase in subcutis of the thigh (+54%; p = 0.048) and abdomen (+68%; p = 0.009). Adults showed an age-related decrease in dermal (p = 0.021) and subcutis (p = 0.009) thicknesses. Pubertal girls had a thicker subcutis than pubertal boys in both thigh (16.7 vs 7.5 mm; p&lt;0.0001) and abdomen (16.7 vs 8.8 mm; p&lt;0.0001). Men had greater thigh dermal thickness than women (1.89 vs 1.65 mm; p = 0.003), while the subcutis was thicker in women in thigh (21.3 vs 17.9 mm; p = 0.012) and abdomen (17.7 vs 9.8 mm; p&lt;0.0001). In boys, men, and women, both dermis and subcutis were thicker on the abdomen compared to thigh; in girls this was only so for dermal thickness. In both children and adults, the skin (dermis and subcutis) became steadily thicker with increasing BMI (p&lt;0.0001).</p><p>Conclusions</p><p>Skin thickness is affected by age, pubertal status, gender, BMI, and anatomical site. Such differences may be important when considering appropriate sites for dermal/subcutaneous injections and other transdermal delivery systems.</p></div

Skin thickness in boys (blue bars) and girls (red bars), according to pubertal status.

<p>****p&lt;0.0001.</p

The association of BMI SDS and BMI with thickness of skin layers in children (n = 103) and adults (n = 140), respectively.

<p>Data for females are in red and for males in blue.</p