Maree Bilandzic

Isolation and Characterization of Tumor Cells from the Ascites of Ovarian Cancer Patients: Molecular Phenotype of Chemoresistant Ovarian Tumors

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12–14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

Betaglycan: a multifunctional accessory.

Betaglycan is a co-receptor for the TGFβ superfamily, particularly important in establishing the potency of its ligands on their target cells. In recent years, new insights have been gained into the structure and function of betaglycan, expanding its role from that of a simple co-receptor to include additional ligand-dependent and ligand-independent roles. This review focuses on recent advances in the betaglycan field, with a particular emphasis on its newly discovered actions in mediating the trafficking of TGFβ superfamily receptors and as a determinant of the functional output of TGFβ superfamily signalling. In addition, this review encompasses a discussion of the emerging roles of the betaglycan/inhibin pathway in reproductive cancers and disease.

Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells

Betaglycan is a type III TGFbeta receptor that modulates cellular sensitivity to inhibins and TGFbeta. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFbeta and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells, decreased cellular invasion through Matrigel, and inhibited wound healing. Expression of mutant forms of betaglycan that are defective in TGFbeta and/or inhibin binding in each GCT cell line revealed that the inhibitory effects of betaglycan on wound healing were most strongly linked to the inhibin-binding region of betaglycan. Furthermore, knockdown of INHA mRNA expression abrogated the betaglycan-mediated inhibition of wound healing and invasion, whereas both INHA silencing and TGFbeta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. These data suggest that loss of betaglycan contributes to the pathogenesis of GCTs.

Increasing Intracellular Bioavailable Copper Selectively Targets Prostate Cancer Cells

The therapeutic efficacy of two bis(thiosemicarbazonato) copper complexes, glyoxalbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)], for the treatment of prostate cancer was assessed in cell culture and animal models. Distinctively, copper dissociates intracellularly from Cu(II)(gtsm) but is retained by Cu(II)(atsm). We further demonstrated that intracellular H2gtsm [reduced Cu(II)(gtsm)] continues to redistribute copper into a bioavailable (exchangeable) pool. Both Cu(II)(gtsm) and Cu(II)(atsm) selectively kill transformed (hyperplastic and carcinoma) prostate cell lines but, importantly, do not affect the viability of primary prostate epithelial cells. Increasing extracellular copper concentrations enhanced the therapeutic capacity of both Cu(II)(gtsm) and Cu(II)(atsm), and their ligands (H2gtsm and H2atsm) were toxic only toward cancerous prostate cells when combined with copper. Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with Cu(II)(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (∼70%) and severity (grade), while treatment with Cu(II)(atsm) (30 mg/kg) was ineffective at the given dose. However, Cu(II)(gtsm) caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention. Mechanistically, we demonstrated that Cu(II)(gtsm) inhibits proteasomal chymotrypsin-like activity, a feature further established as being common to copper-ionophores that increase intracellular bioavailable copper. We have demonstrated that increasing intracellular bioavailable copper can selectively kill cancerous prostate cells in vitro and in vivo and have revealed the potential for bis(thiosemicarbazone) copper complexes to be developed as therapeutics for prostate cancer.

Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success

STUDY QUESTION Do human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity? SUMMARY ANSWER Blastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not. WHAT IS KNOWN ALREADY Human preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium. STUDY DESIGN, SIZE, DURATION We used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase. PARTICIPANTS/MATERIALS, SETTING, METHODS We used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3). MAIN RESULTS AND THE ROLE OF CHANCE Blastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05). LIMITATIONS, REASONS FOR CAUTION This is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse. WIDER IMPLICATIONS OF THE FINDINGS This new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success. STUDY FUNDING/COMPETING INTEREST This study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.

Betaglycan Alters NFκB-TGFβ2 Cross Talk to Reduce Survival of Human Granulosa Tumor Cells

The molecular pathways controlling granulosa cell tumor (GCT) survival are poorly understood. In many cell types, nuclear factor-κB (NFκB) and TGFβ coordinately regulate cell survival to maintain tissue homeostasis. Because GCT cell lines exhibit constitutively activated NFκB, we hypothesized that NFκB blocks TGFβ-mediated cell death in GCT cells. To test this hypothesis, we used the human GCT cell line KGN, which exhibits loss of betaglycan, a TGFβ co-receptor. After inhibition of NFκB in KGN cells, re-expression of betaglycan resulted in a decrease in cell viability, which was further decreased by TGFβ2. Intriguingly, TGFβ2 increased NFκB reporter activity in control cells, but betaglycan expression suppressed both basal and TGFβ2-stimulated NFκB activity. Chemical inhibition of Mothers against decapentaplegic homolog 2/3 (SMAD2/3) signaling or SMAD2/3 gene silencing revealed that both SMADs contributed to cell survival. Furthermore, inhibiting NFκB activity resulted in a specific reduction in SMAD3 expression. Conversely, overexpression of SMAD3 increased basal NFκB activity and countered betaglycan-mediated suppression of NFκB activity. Finally, ERK1/2 activation emerged as the point of convergence of NFκB, SMAD3, and TGFβ2/betaglycan governance of GCT cell viability. Key findings in KGN cells were reproduced in a second GCT cell line, COV434. Collectively, our data establish that both SMAD2/3 and NFκB signaling pathways support GCT cell viability and suggest the existence of a positive feedback loop between NFκB and SMAD3 signaling in late-stage GCT. Furthermore, our data suggest that loss of betaglycan during tumor progression in GCT alters the functional outcomes generated by NFκB and TGFβ pathway cross talk.

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Reprint of: Betaglycan: a multifunctional accessory.

Betaglycan is a co-receptor for the TGFβ superfamily, particularly important in establishing the potency of its ligands on their target cells. In recent years, new insights have been gained into the structure and function of betaglycan, expanding its role from that of a simple co-receptor to include additional ligand-dependent and ligand-independent roles. This review focuses on recent advances in the betaglycan field, with a particular emphasis on its newly discovered actions in mediating the trafficking of TGFβ superfamily receptors and as a determinant of the functional output of TGFβ superfamily signalling. In addition, this review encompasses a discussion of the emerging roles of the betaglycan/inhibin pathway in reproductive cancers and disease.

Betaglycan blocks metastatic behaviors in human granulosa cell tumors by suppressing NFκB-mediated induction of MMP2

Metastatic ovarian granulosa cell tumors (GCT) exhibit loss of betaglycan. Here we test the hypothesis that betaglycan blocks GCT metastasis by suppressing NFκB/TGFβ2-induced matrix metalloprotinease-2 (MMP2). Human GCT and a human GCT cell model demonstrated prominent MMP2 expression, which was dependent on NFκB activity and stimulated by TGFβ2 in an NFκB-dependent manner. Betaglycan suppressed both basal and TGFβ2-induced MMP2 expression and countered metastatic behaviors of GCT cells in non-adherent spheroid culture and in vivo xenograft models of metastasis. These data suggest that NFκB/TGFβ2 promotes, and betaglycan impedes, the early stages of GCT metastasis, when tumor cells first invade the peritoneum.

Discovery and Validation of Novel Protein Biomarkers in Ovarian Cancer Patient Urine

For the vast majority of ovarian cancer patients, optimal surgical debulking remains a key prognostic factor associated with improved survival. A standardized, biomarker‐based test, to preoperatively discriminate benign from malignant disease and inform appropriate patient triage, is highly desirable. However, no fit‐for‐purpose biomarkers have yet been identified.