Linking genomic reorganization to tumor initiation via the giant cell cycle.

To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse.

Gankyrin facilitates follicle-stimulating hormone-driven ovarian cancer cell proliferation through the PI3K/AKT/HIF-1α/cyclin D1 pathway.

Gankyrin is a regulatory subunit of the 26kD proteasome complex. As a novel oncoprotein, gankyrin is expressed aberrantly in cancers from several different sites and has been shown to contribute to oncogenesis in endometrial and cervical carcinomas. Neither gankyrin's contribution to the development of epithelial ovarian cancer nor its interaction with follicle-stimulating hormone (FSH)-driven proliferation in ovarian cancer has been studied. Here we have found that gankyrin is overexpressed in ovarian cancers compared with benign ovarian cystadenomas and that gankyrin regulates FSH upregulation of cyclin D1. Importantly, gankyrin regulates PI3K/AKT signaling by downregulating PTEN. Prolonged AKT activation by FSH stimulation of the FSH receptor (FSHR) promotes gankyrin expression, which, in turn, enhances AKT activation by inhibiting PTEN. Overexpression of gankyrin decreases hypoxia inducible factor-1α (HIF-1α) protein levels, but has little effect on HIF-1α mRNA levels, which could be attributed to gankyrin mediating HIF-1α protein stability via the ubiquitin-proteasome pathway. Reduction in HIF-1α protein stability led to attenuation of the binding with cyclin D1 promoter, resulted in abolishment of the negative regulation of cyclin D1 by HIF-1α, which promotes proliferation of ovarian cancer cells. Our results document that gankyrin regulates HIF-1α protein stability and cyclin D1 expression, ultimately mediating FSH-driven ovarian cancer cell proliferation.

ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts.

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.

ARHI (DIRAS3) induces autophagy in ovarian cancer cells by downregulating the epidermal growth factor receptor, inhibiting PI3K and Ras/MAP signaling and activating the FOXo3a-mediated induction of Rab7.

The process of autophagy has been described in detail at the molecular level in normal cells, but less is known of its regulation in cancer cells. Aplasia Ras homolog member I (ARHI; DIRAS3) is an imprinted tumor suppressor gene that is downregulated in multiple malignancies including ovarian cancer. Re-expression of ARHI slows proliferation, inhibits motility, induces autophagy and produces tumor dormancy. Our previous studies have implicated autophagy in the survival of dormant ovarian cancer cells and have shown that ARHI is required for autophagy induced by starvation or rapamycin treatment. Re-expression of ARHI in ovarian cancer cells blocks signaling through the PI3K and Ras/MAP pathways, which, in turn, downregulates mTOR and initiates autophagy. Here we show that ARHI is required for autophagy-meditated cancer cell arrest and ARHI inhibits signaling through PI3K/AKT and Ras/MAP by enhancing internalization and degradation of the epidermal growth factor receptor. ARHI-mediated downregulation of PI3K/AKT and Ras/ERK signaling also decreases phosphorylation of FOXo3a, which sequesters this transcription factor in the nucleus. Nuclear retention of FOXo3a induces ATG4 and MAP-LC3-I, required for maturation of autophagosomes, and also increases the expression of Rab7, required for fusion of autophagosomes with lysosomes. Following the knockdown of FOXo3a or Rab7, autophagolysosome formation was observed but was markedly inhibited, resulting in numerous enlarged autophagosomes. ARHI expression correlates with LC3 expression and FOXo3a nuclear localization in surgical specimens of ovarian cancer. Thus, ARHI contributes to the induction of autophagy through multiple mechanisms in ovarian cancer cells.

Cyclin G1 regulates the outcome of taxane-induced mitotic checkpoint arrest.

Anti-mitotic chemotherapeutic agents such as taxanes activate the spindle assembly checkpoint (SAC) to arrest anaphase onset, but taxane-exposed cells eventually undergo slippage to exit mitosis. The therapeutic efficacy of taxanes depends on whether slippage after SAC arrest culminates in continued cell survival, or in death by apoptosis. However, the mechanisms that determine these outcomes remain unclear. Here, we identify a novel role for cyclin G1 (CCNG1), an atypical cyclin. Increased CCNG1 expression accompanies paclitaxel-induced, SAC-mediated mitotic arrest, independent of p53 integrity or signaling through the SAC component, BUBR1. CCNG1 overexpression promotes cell survival after paclitaxel exposure. Conversely, CCNG1 depletion by RNA interference delays slippage and enhances paclitaxel-induced apoptosis. Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds. Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.

The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways.

Ovarian cancers migrate and metastasize over the surface of the peritoneal cavity. Consequently, dysregulation of mechanisms that limit cell migration may be particularly important in the pathogenesis of the disease. ARHI is an imprinted tumor-suppressor gene that is downregulated in >60% of ovarian cancers, and its loss is associated with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast to Ras, ARHI inhibits cell growth, but whether it also regulates cell motility has not been studied previously. Here we report that re-expression of ARHI decreases the motility of IL-6- and epidermal growth factor (EGF)-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI binds to and sequesters Stat3 in the cytoplasm, preventing its translocation to the nucleus and localization in focal adhesion complexes. Stat3 siRNA or the JAK2 inhibitor AG490 produced similar inhibition of motility. However, the combination of ARHI expression with Stat3 knockdown or inhibition produced greatest inhibition in ovarian cancer cell migration, consistent with Stat3-dependent and Stat3-independent mechanisms. Consistent with two distinct signaling pathways, knockdown of Stat3 selectively inhibited IL-6-stimulated migration, whereas knockdown of focal adhesion kinase (FAK) preferentially inhibited EGF-stimulated migration. In EGF-stimulated ovarian cancer cells, re-expression of ARHI inhibited FAK(Y397) and Src(Y416) phosphorylation, disrupted focal adhesions, and blocked FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI also disrupted the formation of actin stress fibers in a FAK- and RhoA-dependent manner. Thus, ARHI has a critical and previously uncharacterized role in the regulation of ovarian cancer cell migration, exerting inhibitory effects on two distinct signaling pathways.

Molecular approaches to personalizing management of ovarian cancer.

Cytoreductive surgery and empirical combination chemotherapy have improved 5-year survival for ovarian cancer patients, but have not increased the overall rate of cure. Poor outcomes relate, at least in part, to late diagnosis and to the persistence of dormant ovarian cancer cells that have resisted conventional drugs. Increased understanding of the molecular, cellular and clinical biology of ovarian cancer must be translated into personalized therapy with conventional and targeted agents as well as personalized detection of high-grade cancers in early stages. Different strategies will be required to treat low-grade and high-grade serous cancers as well as other histotypes. Activating mutations of Ras and Raf can be targeted in low-grade cancers. Activation of the PI3K pathway-PI3Kness-and inactivation of BRCA function-BRCAness-can be targeted in high-grade lesions. Inhibition of multiple pathways will be required. Sensitivity of primary cancers to paclitaxel and platinum can be modulated by inhibiting kinases and other molecules that regulate the cell cycle. Dormant ovarian cancer cells may depend upon autophagy, cytokines and growth factors for survival. Early detection can utilize two stage strategies where rising serum biomarker levels prompt imaging in a small fraction of women. Screening can be personalized by taking into account each woman's baseline biomarker levels.

Clinical application of oxaliplatin in epithelial ovarian cancer.

Platinum remains the most active drug class in ovarian cancer treatment; however, new single-agent and combination therapies are needed to improve the clinical outcome of ovarian cancer therapies. Oxaliplatin, a third-generation platinum derivative, has shown effective antitumor activity and a favorable toxicity profile in epithelial ovarian cancer. Preclinical evidence of the synergistic cytotoxic effect of oxaliplatin in combination with several other chemotherapeutic agents and clinical evidence of the absence of any dose-limiting hematologic toxicity associated with this agent have made oxaliplatin an attractive compound for combination agent therapy. This article reviews the current status of the clinical application of oxaliplatin alone and in a combination regimen in epithelial ovarian cancer treatment.

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy.

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.

New tumor markers: CA125 and beyond.

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

Analysis of 96 patients with advanced ovarian carcinoma treated with high-dose chemotherapy and autologous stem cell transplantation.

The purpose of this study was to identify characteristics significant to survival and progression-free survival in patients with advanced ovarian cancer receiving high-dose chemotherapy. In all, 96 patients received autologous stem cell transplantation. Regimens included paclitaxel with carboplatin (PC), topotecan, melphalan, cyclophosphamide (TMC) and cyclophosphamide, BCNU, thiotepa (CBT). At the time of transplantation, 43% of patients were in clinical CR, 34% were in clinical PR, 18% had progressive disease and 5% had stable disease. There were no treatment-related deaths. The 6-year survival by Kaplan-Meier was 38%. For patients who received transplantation for remission consolidation, the 6-year survival was 53% with a PFS of 29%. On univariate analysis, the CBT regimen, clear cell histology and disease status other than CR prior to treatment were statistically significant adverse prognostic factors. This analysis has demonstrated that patients in clinical remission are most likely to benefit from autologous transplantation, with the exception of patients with clear cell histology. The TMC combination appeared to be superior to the PC and CBT combinations. Comparative studies of different consolidation approaches will be necessary to determine if autologous transplantation is the preferred treatment for this patient population.

Targeted therapy for epithelial ovarian cancer: current status and future prospects.

Despite advances in surgery and chemotherapy, less than 20% of patients with stage III or IV ovarian cancer survive long-term. In the past, cytotoxic regimens have been developed empirically, combining active agents at maximally tolerated doses, often without a clear rationale for their interaction. Advances in understanding the biology of ovarian cancer have identified multiple molecular targets that differ in normal and malignant cells. Targets include cell cycle regulators, growth factor receptors, signal transduction pathways, molecules that confer drug resistance, and angiogenic mechanisms. A number of targeted agents have entered clinical trials. Small molecular weight inhibitors, monoclonal antibodies, and antisense and gene therapy are all being evaluated alone and in combination with cytotoxic drugs. In contrast to earlier studies, the impact of each agent on the designated target can be assessed and agents can be matched to the genotype and phenotype of malignant and normal cells. In the long run, this should facilitate individualization of more effective, less toxic therapy for women with ovarian cancer.

Current research and treatment for epithelial ovarian cancer. A Position Paper from the Helene Harris Memorial Trust.

In March 2003, an international mulltidisciplinary group of scientists and clinicians with a specific interest in ovarian cancer met for 4 days to discuss research into and treatment of this challenging disease. Under the headings of molecular genetics, molecular biology, the biology of ovarian cancer, old therapies, new targets and the early detection of the disease, this Position Paper summarises the presentations and discussion from the 9th Biennial Helene Harris Memorial Trust Forum on Ovarian Cancer. In particular, we highlight the potential of international collaborations in translating laboratory science into useful clinical interventions.

Heregulin-induced apoptosis.

Heregulins (HRGs) are a group of polypeptide factors that are encoded by four different HRG genes that can express multiple isoforms through alternate RNA splicing. A number of HRG isoforms possess both growth stimulatory and growth inhibitory functions that are necessary for their important role in the development and maintenance of the heart, nervous system and epithelial cells in multiple organs including the breast. Growth inhibition by HRG relates to its ability to induce apoptosis, differentiation, and cell cycle G(2) arrest. Current studies suggest that HRGs can induce a unique form of apoptosis. In this article, we review recent progress in characterizing and understanding HRG-induced apoptosis. Particular attention has been given to: (1). the activation of caspases-7 and -9; (2). the role of the anti-apoptotic Bcl-2 protein; and (3). the signaling molecules and pathways that regulate HRG-induced apoptosis, including the p38, JNK, mTOR kinase, and PKC alpha kinase.

Immunoadsorption of sensitized kidney transplant candidates immediately prior to surgery.

Patients with anti-human leucocyte antigen (HLA) antibodies from previous transplantation, blood transfusion are highly sensitized and at risk to hyperacute renal graft loss. As these antibodies are identified to be of pathogenic importance, an effective removal may allow successful transplantation. Six 'high risk patients' [panel-reactive antibodies (PRA) >30% or retransplanted patients with an acutely rejected first graft within 6 months from surgery] were treated by protein A immunoadsorption (IA) immediately prior to transplantation. We treated the calculated plasma volume one to three times prior to surgery: mean 4600 mL (range 2100-10 200 mL). After transplantation we repeated the sessions according to antibody (Ab) recurrence, graft function and signs of rejection. The panel reactive Ab were reduced from mean 65% pre-IA (range 35-85) to lowest 15% (range 0-55). After the course they reappeared to 30% (range 0-90). Five of the six patients had no clinical signs of vascular rejection. At a follow-up of mean 54 months (+/-14) four grafts still function with a mean serum creatinine of 172 micromol/L (+/-57). Protein A IA is a safe and effective adjunct in the treatment of highly sensitized patients awaiting renal transplantation. The treatment immediately prior to operation can prevent hyperacute rejection and increases the graft survival in these patients.

OVX1, macrophage-colony stimulating factor, and CA-125-II as tumor markers for epithelial ovarian carcinoma: a critical appraisal.

BACKGROUND: Ovarian carcinoma remains the leading cause of death from gynecologic malignancy in Australia, the Netherlands, and the United States. CA-125-II, the most widely used serum marker, has limited sensitivity and specificity for detecting small-volume, early-stage disease. Therefore, a panel of three serum tumor markers-OVX1, CA-125-II, and macrophage-colony stimulating factor (M-CSF)-has been used to evaluate the sensitivity and specificity of multiple markers for the detection of early-stage ovarian carcinoma. METHODS: Preoperative serum levels of OVX1, CA-125-II, and M-CSF were measured in 281 patients with primary ovarian epithelial tumors of different histotypes. Among these tumors, 175 were malignant, 29 were of borderline malignancy, and 77 were benign. The three markers also were measured in sera from 117 apparently healthy women. Marker levels were considered abnormal at CA-125-II > 35 U/mL, OVX1 > 7.2 U/mL, and M-CSF > 3.5 ng/mL. RESULTS: Among 175 women with malignant ovarian tumors, at least one of the three serum markers was elevated in 85%, whereas CA-125-II was elevated in 80% (P = 0.008). In 58 patients with Stage I ovarian carcinoma, at least one of the three serum markers was elevated in 76%, whereas CA-125 levels were elevated in 66% (P = 0.04). For patients with borderline and benign tumors, a combination of the three antigens had slightly higher sensitivity compared with CA-125-II, but the differences were not statistically significant. Among 117 apparently healthy women, CA-125-II was elevated in 4%, and one of the three markers was positive in 17%. CONCLUSIONS: The sensitivity of a combination of three serum markers was significantly greater than the sensitivity of the CA-125-II assay alone in patients with primary ovarian epithelial tumors of different histotypes. This was true for all stages, including early-stage, potentially curable disease. When used as single markers, however, only the CA-125-II assay could distinguish invasive Stage I tumors from apparently healthy women.