Yudith Ramos Valdes

Modulation of AKT activity is associated with reversible dormancy in ascites-derived epithelial ovarian cancer spheroids

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.

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.

TGFβ signaling regulates epithelial–mesenchymal plasticity in ovarian cancer ascites-derived spheroids

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease.

BMP signalling controls the malignant potential of ascites-derived human epithelial ovarian cancer spheroids via AKT kinase activation

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 ALK3QD receptor expression causes the formation of smaller, more loosely-aggregated spheroids. Additionally, ALK3QD-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.

Evidence for Differential Viral Oncolytic Efficacy in an In Vitro Model of Epithelial Ovarian Cancer Metastasis

Epithelial ovarian cancer is unique among most carcinomas in that metastasis occurs by direct dissemination of malignant cells traversing throughout the intraperitoneal fluid. Accordingly, we test new therapeutic strategies using an in vitro three-dimensional spheroid suspension culture model that mimics key steps of this metastatic process. In the present study, we sought to uncover the differential oncolytic efficacy among three different viruses—Myxoma virus, double-deleted vaccinia virus, and Maraba virus—using three ovarian cancer cell lines in our metastasis model system. Herein, we demonstrate that Maraba virus effectively infects, replicates, and kills epithelial ovarian cancer (EOC) cells in proliferating adherent cells and with slightly slower kinetics in tumor spheroids. Myxoma virus and vaccinia viruses infect and kill adherent cells to a much lesser extent than Maraba virus, and their oncolytic potential is almost completely attenuated in spheroids. Myxoma virus and vaccinia are able to infect and spread throughout spheroids, but are blocked in the final stages of the lytic cycle, and oncolytic-mediated cell killing is reactivated upon spheroid reattachment. Alternatively, Maraba virus has a remarkably reduced ability to initially enter spheroid cells, yet rapidly infects and spreads throughout spheroids generating significant cell killing effects. We show that low-density lipoprotein receptor expression in ovarian cancer spheroids is reduced and this controls efficient Maraba virus binding and entry into infected cells. Taken together, these results are the first to implicate the potential impact of differential viral oncolytic properties at key steps of ovarian cancer metastasis.

Spatial and temporal epithelial ovarian cancer cell heterogeneity impacts Maraba virus oncolytic potential

BackgroundEpithelial ovarian cancer exhibits extensive interpatient and intratumoral heterogeneity, which can hinder successful treatment strategies. Herein, we investigated the efficacy of an emerging oncolytic, Maraba virus (MRBV), in an in vitro model of ovarian tumour heterogeneity.MethodsFour ovarian high-grade serous cancer (HGSC) cell lines were isolated and established from a single patient at four points during disease progression. Limiting-dilution subcloning generated seven additional subclone lines to assess intratumoral heterogeneity. MRBV entry and oncolytic efficacy were assessed among all 11 cell lines. Low-density receptor (LDLR) expression, conditioned media treatments and co-cultures were performed to determine factors impacting MRBV oncolysis.ResultsTemporal and intratumoral heterogeneity identified two subpopulations of cells: one that was highly sensitive to MRBV, and another set which exhibited 1000-fold reduced susceptibility to MRBV-mediated oncolysis. We explored both intracellular and extracellular mechanisms influencing sensitivity to MRBV and identified that LDLR can partially mediate MRBV infection. LDLR expression, however, was not the singular determinant of sensitivity to MRBV among the HGSC cell lines and subclones. We verified that there were no apparent extracellular factors, such as type I interferon responses, contributing to MRBV resistance. However, direct cell-cell contact by co-culture of MRBV-resistant subclones with sensitive cells restored virus infection and oncolytic killing of mixed population.ConclusionsOur data is the first to demonstrate differential efficacy of an oncolytic virus in the context of both spatial and temporal heterogeneity of HGSC cells and to evaluate whether it will constitute a barrier to effective viral oncolytic therapy.

Abstract B51: Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells.

Aberrant cell metabolism is rapidly establishing itself as a critical hallmark of human malignancies. Cancer cells are faced with huge metabolic demands to support rapid tumor growth, yet are commonly starved for nutrients. In response, cancer cells hijack alternative signaling pathways during these times of energy and metabolic stress to sustain viability. Metastatic epithelial ovarian cancer (EOC) cells are faced with additional stressors during transcoelomic spread, such as detachment from a matrix substratum and inhospitable conditions in peritoneal fluid. However, EOC cells have a natural ability to aggregate when in suspension to form multicellular aggregates, or spheroids, which supports a survival advantage for cells when transiting the peritoneal space during metastatic progression. Therefore, we utilize an in vitro spheroid culture model system to investigate signaling pathways altered in EOC cells that may be implicated in ovarian cancer pathobiology and promote metastasis. For example, we have shown that spheroid formation induces cellular quiescence and autophagy, two disparate processes which promote EOC cell survival and resistance to platinum-based chemotherapeutics. In addition, we discovered that EOC spheroids have significantly reduced mitochondrial activity and ATP levels compared with matched proliferating adherent cells. Liver kinase B1 (LKB1) acts as a chief responder to intracellular stress due to reduced energy and nutrients by eliciting general growth suppression during these starvation-like conditions. Thus, we hypothesized that LKB1 activity is increased in EOC spheroids to promote tumor cell dormancy and cell survival. Although the STK11 gene encoding LKB1 is heterozygously deleted in 84% of serous ovarian tumors, we demonstrate that almost all ovarian tumor cells and established EOC cell lines retain intact LKB1 expression. In fact, LKB1 protein expression increases when EOC cells form spheroids and this coincides with induced serine-428 phosphorylation, a modification that is required for LKB1 growth suppressive activity. To address the potential functional requirement of LKB1 in EOC spheroids, we first performed transient knockdown of STK11 to block LKB1 expression. Indeed, reduced LKB1 renders spheroids susceptible to cell death and increases sensitivity to carboplatin. Next, we utilized Cas9-mediated genome editing of the STK11 locus to completely ablate LKB1 expression in HEYA8 and OVCAR8 cells. STK11 -knockout HEYA8 and OVCAR8 cells yielded significantly decreased spheroid number and viability compared with parental cell lines. In a reciprocal fashion, forced re-expression of LKB1 in CaOV3 and TOV21G cells—two EOC cell lines which harbor inactivating STK11 mutations—reduces cell growth and colony formation in proliferating adherent culture. Proliferation and dispersion of CaOV3-LKB1 and TOV21G-LKB1 cells from re-attached spheroids are also reduced. Taken together, LKB1 has growth suppressive effects in EOC cells, yet it serves the additional purpose to promote cell survival in spheroids. The canonical target of LKB1 is AMP-activated protein kinase (AMPK), which is thought to elicit the majority of LKB1 growth suppressive effects during stress metabolism signaling. As expected, AMPK phosphorylation at threonine-172 is also induced upon spheroid formation. Using STK11 -knockout EOC cells, however, we demonstrate that LKB1 is not required to phosphorylate AMPK in spheroids; more importantly, knockdown of PRKAA1 encoding the catalytic alpha-subunit of AMPK has no effect on spheroid cell viability. Thus, our results suggest that LKB1 utilizes alternative mechanisms to regulate the dormancy phenotype in EOC spheroids. Future work will entail direct experiments testing whether LKB1-mediated stress metabolism signaling has the potential to promote EOC metastasis and recurrence of chemo-resistant disease. Citation Format: Trevor G. Shepherd, Yudith Ramos Valdes, Teresa Peart, Meera Shah, Gabriel E. DiMattia. Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B51.