Loss of LKB1-NUAK1 signalling enhances NF-κB activity in a spheroid model of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.

Transcriptional heterogeneity of stemness phenotypes in the ovarian epithelium.

The ovarian surface epithelium (OSE) is a monolayer of epithelial cells surrounding the ovary that ruptures during each ovulation to allow release of the oocyte. This wound is quickly repaired, but mechanisms promoting repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, but their involvement in OSE is unclear. We show that traits associated with stem cells can be increased following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. We find that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not be associated with a single, distinct population, but rather is a heterogeneous cell state that may emerge from diverse environmental cues. These findings support that the OSE may not require distinct stem cells for long-term maintenance, and may instead achieve this through transient dedifferentiation into a stem-like state.

Activated CAMKKß-AMPK signaling promotes autophagy in a spheroid model of ovarian tumour metastasis.

BACKGROUND: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. RESULTS: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKß inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. CONCLUSIONS: Our results support the premise that CAMKKß-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in spheroids.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

COX2 is induced in the ovarian epithelium during ovulatory wound repair and promotes cell survival†.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation, the wound is repaired, however, this process is poorly understood. In epithelial tissues, wound repair is mediated by an epithelial-to-mesenchymal transition (EMT). Transforming Growth Factor Beta-1 (TGFß1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We, therefore, hypothesized that TGFß1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFß1 induced an EMT mediated by TGFßRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFß1 and, when overexpressed, was shown to increase OSE cell migration. A polymerase chain reaction array of TGFß signaling targets determined Cyclooxygenase-2 (Cox2) to be most highly induced by TGFß1. Constitutive Cox2 expression modestly increased migration and robustly enhanced cell survival, under stress conditions similar to those observed during wound repair. The increase in Snail and Cox2 expression with TGFß1 was reproduced in human OSE cultures, suggesting these responses are conserved between mouse and human. Finally, the induction of Cox2 expression in OSE cells during ovulatory wound repair was shown in vivo, suggesting TGFß1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFß1 promotes ovulatory wound repair by induction of an EMT and activation of a COX2-mediated pro-survival pathway. Understanding ovulatory wound repair may give insight into why ovulation is the primary non-hereditary risk factor for ovarian cancer.

PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state.

Recent studies have provided evidence that the secretory cells of the fallopian tube (oviduct) are a probable origin for high-grade serous ovarian carcinoma. In addition to secretory cells, the fallopian tube epithelium consists of ciliated cells and CD44+ undifferentiated stem-like cells. Loss of PAX2 expression is recognized as an early event in epithelial transformation, but the specific role of PAX2 in this transition is unknown. The aim of this study was to define the role of PAX2 in oviductal epithelial (OVE) cells and its response to transforming growth factor ß1 (TGFß), characterizing specifically its potential involvement in regulating stem cell-like behaviors that may contribute to formation of cancer-initiating cells. Treatment of primary cultures of mouse OVE cells with TGFß induced an epithelial-mesenchymal transition (EMT) associated with decreased expression of PAX2 and an increase in the fraction of cells expressing CD44. PAX2 knockdown in OVE cells and overexpression in ovarian epithelial cells confirmed that PAX2 inhibits stem cell characteristics and regulates the degree of epithelial differentiation of OVE cells. These results suggest that loss of PAX2, as occurs in serous tubal intraepithelial carcinomas, may shift secretory cells to a more mesenchymal phenotype associated with stem-like features.

A new spontaneously transformed syngeneic model of high-grade serous ovarian cancer with a tumor-initiating cell population.

Improving screening and treatment options for patients with epithelial ovarian cancer has been a major challenge in cancer research. Development of novel diagnostic and therapeutic approaches, particularly for the most common subtype, high-grade serous ovarian cancer (HGSC), has been hampered by controversies over the origin of the disease and a lack of spontaneous HGSC models to resolve this controversy. Over long-term culture in our laboratory, an ovarian surface epithelial (OSE) cell line spontaneously transformed OSE (STOSE). The objective of this study was to determine if the STOSE cell line is a good model of HGSC. STOSE cells grow faster than early passage parental M0505 cells with a doubling time of 13 and 48 h, respectively. STOSE cells form colonies in soft agar, an activity for which M0505 cells have negligible capacity. Microarray analysis identified 1755 down-regulated genes and 1203 up-regulated genes in STOSE compared to M0505 cells, many associated with aberrant Wnt/ß-catenin and Nf-κB signaling. Upregulation of Ccnd1 and loss of Cdkn2a in STOSE tumors is consistent with changes identified in human ovarian cancers by The Cancer Genome Atlas. Intraperitoneal injection of STOSE cells into severe combined immunodeficient and syngeneic FVB/N mice produced cytokeratin+, WT1+, inhibin-, and PAX8+ tumors, a histotype resembling human HGSC. Based on evidence that a SCA1+ stem cell-like population exists in M0505 cells, we examined a subpopulation of SCA1+ cells that is present in STOSE cells. Compared to SCA1- cells, SCA1+ STOSE cells have increased colony-forming capacity and form palpable tumors 8 days faster after intrabursal injection into FVB/N mice. This study has identified the STOSE cells as the first spontaneous murine model of HGSC and provides evidence for the OSE as a possible origin of HGSC. Furthermore, this model provides a novel opportunity to study how normal stem-like OSE cells may transform into tumor-initiating cells.

The mouse ovarian surface epithelium contains a population of LY6A (SCA-1) expressing progenitor cells that are regulated by ovulation-associated factors.

The ovarian surface epithelium, a single layer of poorly differentiated epithelial cells, covers the surface of the ovary and is ruptured during ovulation. Little is known about the changes that occur in this layer before or during ovulation, and even less is known about the regenerative processes that occur after the surface is ruptured to release a mature oocyte. Recently, a population of mouse ovarian surface epithelial (MOSE) cells that exhibit progenitor/stem cell characteristics has been identified, though neither a genetic marker nor how these cells are regulated has been determined. We have identified a defined population of MOSE cells with progenitor cell characteristics that express the stem cell marker lymphocyte antigen 6 complex, locus A (LY6A; also known as stem cell antigen-1 [SCA-1]). By testing the effect of factors found in the follicular fluid at ovulation on proliferation, sphere formation, and LY6A expression, we have determined that the size of the LY6A-expressing (LY6A+) progenitor cell population is regulated by at least two ovulation-associated factors present in the follicular fluid: transforming growth factor beta 1 and leukemia-inhibitory factor. Our work has identified a population of LY6A+ MOSE progenitor cells on the surface of the ovary that may play a role in ovulatory wound healing.

Conditional inactivation of Brca1, p53 and Rb in mouse ovaries results in the development of leiomyosarcomas.

Epithelial ovarian cancer (EOC) is thought to arise in part from the ovarian surface epithelium (OSE); however, the molecular events underlying this transformation are poorly understood. Germline mutations in the BRCA1 tumor suppressor gene result in a significantly increased risk of developing EOC and a large proportion of sporadic EOCs display some sort of BRCA1 dysfunction. To generate a model in which Brca1-mediated transformation can be studied, we previously inactivated Brca1 alone in murine OSE, which resulted in an increased accumulation of premalignant changes, but no tumor formation. In this study, we examined tumor formation in mice with conditionally expressed alleles of Brca1, p53 and Rb, alone or in combination. Intrabursal injection of adenovirus expressing Cre recombinase to inactivate p53 resulted in tumors in 100% of mice. Tumor progression was accelerated in mice with concomitant inactivation of Brca1 and p53, but not Rb and p53. Immunohistologic analyses classified the tumors as leiomyosarcomas that may be arising from the ovarian bursa. Brca1 inactivation in primary cultures of murine OSE cells led to a suppression of proliferation that could be rescued by concomitant inactivation of p53 and/or Rb. Brca1-deficient OSE cells displayed an increased sensitivity to the DNA damaging agent cisplatin, and this effect could be modulated by inactivation of p53 and/or Rb. These results indicate that Brca1 deficiency can accelerate tumor development and alter the sensitivity of OSE cells to chemotherapeutic agents. Intrabursal delivery of adenovirus intended to alter gene expression in the ovarian surface epithelium may, in some strains of mice, result in more rapid transformation of adjacent cells, resulting in leiomyosarcomas.

Basal and zinc-induced metallothionein in resistance to cadmium, cisplatin, zinc, and tertbutyl hydroperoxide: studies using MT knockout and antisense-downregulated MT in mammalian cells.

Metallothioneins (MTs) mediate resistance to metal and non-metal toxicants. To differentiate the role of MTs from other protective factors, resistance to zinc (Zn), cadmium (Cd), tertbutyl hydroperoxide (tBH), and cisplatin (CDDP) was compared in renal cell lines from wild type (MT-WT) and MT-1/MT-2 knockout (MT-KO) mice. MT-WT cells were more resistant to tBH than MT-KO cells but, unexpectedly, were more sensitive to Zn, Cd, and CDDP. Thus, basal expression of MT conferred resistance to tBH, but not to Cd or CDDP. Pretreatment with Zn increased MT expression and enhanced resistance to Cd and CDDP only in MT-WT cells, indicating a critical role for MT in this form of resistance. By contrast, Zn-pretreatment increased resistance to subsequent Zn exposure, but did not alter resistance to tBH, regardless of MT-status. Therefore, Zn-induced resistance to subsequent exposure to Zn (but not to Cd or CDDP) was mediated by non-MT factors, and neither Zn-induced MT nor other factors affected tBH sensitivity. Furthermore, antisense down-regulation of MT in human HeLa cells reduced basal MT levels and resistance to TBH, but not to Cd or CDDP. Therefore, basal MT alone can mediate resistance to TBH (but not to Cd or CDDP) in mouse and human cells. These data suggest that MT can mediate resistance to toxicants by different mechanisms, some of which correlate with the cellular content of MT protein. Moreover, resistance to some agents (Cd and CDDP) can be enhanced by inducing MT. Resistance to other agents (tBH) requires only basal (non-induced) MT levels.

Metallothionein mediates the level and activity of nuclear factor kappa B in murine fibroblasts.

The zinc-binding protein metallothionein (MT) is associated with resistance to apoptosis. We examined whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor KappaB (NF-KappaB), which is up-regulated under many conditions that lead to elevated MT expression. NF-KappaB protein levels and NF-KappaB-dependent reporter gene activity were examined in clonal MT(+) (MT-WT) and MT(-) (MT-KO) fibroblastic cell lines. The amount of cellular NF-KappaB p65 protein in MT-KO was less than 20% of the amount in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-KappaB p65 mRNA levels, and NF-KappaB p50 subunit and IKappaBalpha protein levels, were unchanged. NF-KappaB activity assessed by expression of a transfected NF-KappaB reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NF-KappaB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-KappaB p65 than did MT-WT cells, despite a lower cellular NF-KappaB level and function, suggesting that metallothionein mediated the specific activity of NF-KappaB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-KappaB p65 (but not IKappaBalpha or NF-KappaB p50), increased NF-KappaB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NF-KappaB.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein.

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.