Downregulation of cell surface CA125/MUC16 induces epithelial-to-mesenchymal transition and restores EGFR signalling in NIH:OVCAR3 ovarian carcinoma cells.

BACKGROUND: Epithelial ovarian cancer (EOC) cells are prone to metastasise throughout the peritoneal cavity. The epithelial-to-mesenchymal transition (EMT) is a necessary step towards metastatic tumour progression. CA125/MUC16 mucin is a high-molecular-weight glycoprotein overexpressed in the majority of serous carcinomas, suggesting a possible role in the pathogenesis of these cancers. METHODS: The role of CA125/MUC16 in EMT was investigated using single-chain antibody-mediated knockdown of cell surface CA125/MUC16 in overexpressing EOC NIH:OVCAR3 cells. RESULTS: CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin). Co-immunoprecipitation experiments revealed that CA125/MUC16 binds to E-cadherin and ß-catenin complexes. The in vitro studies showed disruption of cell-cell junctions, enhanced motility, migration and invasiveness in CA125/MUC16 knockdown cells. Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities. Epidermal growth factor receptor inhibition strongly inhibited the motility of CA125/MUC16 knockdown cells. CONCLUSIONS: Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and ß-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

The inhibition of Bid expression by Akt leads to resistance to TRAIL-induced apoptosis in ovarian cancer cells.

Epithelial ovarian cancer (EOC) cells often show increased activity of the PI3K/Akt pathway. In addition, we have previously shown that EOC ascites induce Akt activation in the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive EOC cell line, CaOV3, leading to TRAIL-mediated apoptosis inhibition. In this study, we investigated the role of Akt in intrinsic resistance to TRAIL, which is common in EOC cells. We report that Akt activation reduces the sensitivity of EOC cells to TRAIL. TRAIL-resistant SKOV3ip1 and COV2 cells were sensitized to TRAIL-induced apoptosis by PI3K or Akt inhibitors although inhibition of PI3K/Akt signaling pathway did not interfere with the recruitment and processing of caspase-8 to the death-inducing signaling complex. Conversely, overexpression of Akt1 in TRAIL-sensitive cells promoted resistance to TRAIL. Although the fact that TRAIL-induced caspase-8 activation was observed in both sensitive and resistant cell lines, Bid cleavage occurred only in sensitive cells or in SKOV3ip1 cells treated with LY294002. Bid expression was low in resistant cells and Akt activation downregulated its expression. Depletion of Bid by siRNA in OVCAR3 cells was associated with a decrease in TRAIL-mediated apoptosis. Overexpression of Bid only in SKOV3ip1 cells enhanced TRAIL-induced apoptosis. Simultaneous blockade of Akt pathway further increased TRAIL-induced apoptosis. Thus, Akt acts upstream of mitochondria and inhibits TRAIL-induced apoptosis by decreasing Bid protein levels and possibly inhibiting its cleavage.

Ovarian cancer ascites protects from TRAIL-induced cell death through alphavbeta5 integrin-mediated focal adhesion kinase and Akt activation.

Interactions between ovarian cancer cells and the surrounding tumor microenvironment are not well characterized. We have earlier shown that ovarian cancer ascites induces Akt activation and protect tumor cells from TRAIL-induced apoptosis. Here, we investigated the mechanism by which ascites activates Akt. The ability of ovarian cancer ascites to activate Akt and inhibit TRAIL-induced cell death and caspase activity was decreased by heat inactivation, but was retained in ascites fractions >5 kDa. The survival promoting activity of ascites was not affected by inhibitors of growth factor receptor including epidermal growth factor receptor (EGFR), VEGFR, FGFR, Her2/neu, and IGF-R1. However, this activity was inhibited by an alphavbeta5 integrin-blocking antibody, but not by blocking antibodies against alphavbeta3, beta1, or beta3 integrins. alphavbeta5 integrin-blocking antibodies also inhibited ascites-induced Akt phosphorylation and c-FLIPs up-regulation. Ovarian cancer ascites induced a rapid phosphorylation of focal adhesion kinase (FAK), which closely correlated with the phosphorylation of Akt overtime. FAK phosphorylation was strongly inhibited by alphavbeta5 integrin-blocking antibodies. Depletion of FAK content by RNA interference was also associated with inhibition of ascites-mediated Akt activation and survival. These results suggest that ovarian cancer ascites induces FAK and Akt activation in an alphavbeta5 integrin-dependent pathway, which confers protection from TRAIL-induced cell death and caspase activation.

Cell detachment modulates TRAIL resistance in ovarian cancer cells by downregulating the phosphatidylinositol 3-kinase/Akt pathway.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a potent inducer of apoptosis but many ovarian cancer cells display intrinsic resistance to TRAIL. The molecular determinants regulating TRAIL sensitivity in these resistant tumor cells are still incompletely understood. We observed that cell detachment enhances TRAIL-induced apoptosis in two TRAIL-resistant ovarian cancer cell lines. This process was accompanied by an increase of caspase activation, which could be blocked by caspase-8 inhibitor IETD. Cell detachment inhibited Akt phosphorylation. Phosphatidylinositol 3-kinase inhibition by LY294002 also enhanced TRAIL-induced apoptosis. Further decreased Akt activity by LY294002 in detached cells translated to increased cell death after TRAIL treatment. Our data indicate that cell detachment enhances TRAIL-induced killing by decreasing Akt activity in TRAIL-resistant ovarian carcinoma cells and suggest that Akt inhibition primes TRAIL-resistant cells to TRAIL-induced apoptosis.

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis.

Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun amino-terminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.

Delivery of herpes simplex thymidine kinase bystander effect by engineered human mesothelial cells for the treatment of ovarian cancer.

BACKGROUND: Resistance to conventional chemotherapy is a major clinical problem for ovarian cancer, and long-term survival for patients with advanced-stage disease is rare. Other therapeutic strategies, such as gene therapy, have been explored but several limitations exist, which include low viral vector transduction efficiency, host immune response to the vector, and vector toxicity. METHODS: We developed a cell-based therapy that exploits human mesothelial cells to deliver anticancer modalities for treatment of ovarian cancer. As a proof of concept, we genetically engineered mesothelial with the herpes simplex virus thymidine kinase/ganciclovir (HSVTK/GCV) system to deliver cytotoxicity to human ovarian cancer cells. This system is well characterized, and has been widely used in different gene-therapy based strategies. RESULTS: Our results demonstrate that HSVTK-modified mesothelial cells are sensitive to GCV killing in vitro and support the HSVTK bystander effect. Engineered mesothelial cells can deliver the HSVTK bystander effect to human ovarian cancer cell-lines, as well as to primary ovarian cancer cells. A significant reduction of tumor growth and prolongation of survival in s.c. and i.p. xenograft mouse models of ovarian cancer are obtained with as little as 1% of HSVTK-expressing mesothelial cells. Intraperitoneal administration of HSVTK-expressing mesothelial cells in an established mouse model of ovarian cancer results in a statistically significant prolonged survival of treated animals. Importantly, distribution studies showed that mesothelial cells localize preferentially to tumor sites. DISCUSSION: Our study demonstrates the feasibility of using a mesothelial cell-based therapy for treatment of ovarian cancer, and suggests that this strategy should be further explored.

Gene therapy to overcome drug resistance in cancer: targeting key regulators of the apoptotic pathway.

A better understanding of the molecular events responsible for the development of drug resistance in cancer cells has emerged in recent years. It is now established that tumor cells can acquire drug resistance by alterations of pathways involved in the regulation of apoptosis and that failure to activate this pathway in cancer cells may confer resistance to chemotherapy. This resistance to drug-induced apoptosis is likely to play an important role in tumors that are refractory to chemotherapy. The identification of points in the apoptotic pathway at which dysregulation occurs opens up new therapeutic opportunities in situations where conventional cytotoxic chemotherapy approaches fail. Although these gene therapy-based strategies are still in their infancy they will likely lead to more effective treatments for human cancers. This review will focus on gene therapy strategies developed to specifically target the apoptotic pathway and how these strategies can affect the sensitivity of tumor cells to chemotherapy.

A role for intracellular immunization in chemosensitization of tumor cells?

Acquired drug resistance represents a major cause of chemotherapy failure in patients with cancer. The characterization of the molecular pathways involved in drug resistance has provided us with new targets to overcome this problem. Many of these target proteins are often overexpressed in human cancers. A number of gene therapy strategies, including antisense oligonucleotides, ribozymes and single-chain antibodies, have been developed to achieve the selective modulation and inhibition of various cellu- lar proteins. Thus, these approaches can be exploited to modulate the resistance phenotype of tumor cells. These gene therapy strategies represent a novel and unique way to enhance the sensitivity of tumor cells to chemotherapeutic drugs. This review will focus on the use of intracellular immunization as a means to modulate the expression of specific genetic determinants involved in the drug resistance phenotype.

An advanced generation of adenoviral vectors selectively enhances gene transfer for ovarian cancer gene therapy approaches.

OBJECTIVE: We hypothesized that incorporation of an integrin binding Arg-Gly-Asp (RGD)-containing peptide to the HI loop of the adenovirus fiber knob would allow enhanced, coxsackie-adenovirus receptor-independent gene transfer by modified Ad vector in the context of ovarian cancer. METHODS: Ovarian cancer cell lines, primary ovarian cancer cells, primary tumor explants, and mesothelial tissue were transfected with luciferase encoding adenovirus (AdCMVLuc) or a genetically modified adenovirus (Ad5lucRGD) which contained an RGD motif within the HI loop of the knob. The luciferase activity was measured and the transduction efficiencies of both viruses were compared. RESULTS: In all established ovarian cell lines and primary tumor cell samples there was dramatically augmented gene transfer observed with the Ad5lucRGD compared to AdCMVLuc. The enhanced gene transfer in ovarian cancer cell lines ranged from 2.5- to 471.6-fold, in ascites samples from 26.1- to 64.0-fold, and in tumor explants from 1.6- to 11.1-fold. Although gene transfer to normal mesothelial tissue was slightly augmented by RGD retargeting, the level of gene transfer was much lower than that seen in ovarian cancer cells. CONCLUSION: This study demonstrates that genetically altered adenoviruses with modified tropism are capable of more efficient gene transfer in the context of ovarian cancer. The higher level of transfer with respect to peritoneal mesothelium can be exploited to enhance the therapeutic index of interventions using adenoviral vectors. Studies are warranted, therefore, to determine the in vivo utility of this targeted vector approach in the context of gene therapeutic strategies for cancer of the ovary.

Interleukin-6 modulated conditionally replicative adenovirus as an antitumor/cytotoxic agent for cancer therapy.

In this study, we report that an interleukin-6 (IL-6)-inducible E1A-substituting activity can be exploited for the production of infectious adenoviral particles during infection with the E1A-deleted adenovirus (Ad) Ad5dl312. The basal level of complementation can be increased by 1.5 log by induction of the HepG2 cells with recombinant human IL-6. Additionally, the IL-6-inducible E1A-substituting activity can complement E1A deletion in other cancer cell lines to render them Ad producer cells on induction with recombinant human IL-6, although the efficiency of complementation varies between cell lines. Ad5dl312 can replicate in, produce cytotoxic effect, and kill human tumor cells without addition of exogenous IL-6 in the context of tumor cells possessing an IL-6 autocrine arc, such as ovarian tumor cells. In contrast, normal human mesothelial cells isolated from normal human peritoneum lining do not support replication of Ad5dl312, even in the presence of exogenous IL-6. These results suggest that Ad5dl312 could be used as a cytotoxic agent to selectively kill tumor cells responsive to or possessing an IL-6 autocrine arc.

High-efficacy thymidine kinase gene transfer to ovarian cancer cell lines mediated by herpes simplex virus type 1 vector.

In this report, a replication-defective herpes simplex virus type 1 (HSV-1) vector has been employed to deliver the Escherichia coli LacZ and HSV thymidine kinase (HSVtk) genes to six human ovarian carcinoma cell lines and the efficacy of gene transfer compared to that of adenoviral vectors in vitro. The transduction efficiency of the LacZ-containing virus TOZ.1 was evaluated qualitatively and quantitatively following infection of the different ovarian cancer cell lines. The therapeutic ability of the HSV-T3 vector, which contains the HSVtk gene, was additionally investigated in comparison to the AdCMVHSVTK. Our results show that HSV-1-mediated gene transfer is quantitatively superior to adenoviral vector in five of the six ovarian cancer cell lines at a 100-fold lower dose in vitro. Our preliminary studies suggest that HSV-1 may be a promising alternative vector for ovarian cancer gene therapy.

Basic fibroblast growth factor enhancement of adenovirus-mediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer.

A number of preclinical and human clinical gene therapy trials using adenoviral vectors have shown that the number of viral particles necessary to give adequate levels of gene transfer can be associated with significant vector-related toxicity. In an effort to reduce the number of adenoviral particles required for a given level of gene transfer, we sought to redirect adenoviral infection via a receptor that is highly expressed on the target cells. By using basic fibroblast growth factor (FGF2) as the targeting ligand, adenovirus-mediated gene transfer to the human ovarian cancer cell line SKOV3.ip1 was significantly enhanced, permitting the transduction of a greater number of target cells to be achieved by a given dose of virus. In a murine model of human ovarian carcinoma, an FGF2-redirected adenoviral vector carrying the gene for herpes simplex virus thymidine kinase (AdCMVHSV-TK) was shown to result in a significant prolongation of survival compared with the same number of particles of unmodified AdCMVHSV-TK. In addition, equivalent survival rates were achieved with a 10-fold lower dose of the FGF2-redirected AdCMVHSV-TK compared with the unmodified vector. To our knowledge, this is the first report demonstrating that strategies to enhance the efficiency of in vivo transduction of adenoviral vectors will be of clinical utility.

Gene therapy for ovarian carcinoma.

Originally conceived and applied for the treatment of inherited monogenetic defects such as adenosine deaminase deficiency and cystic fibrosis, gene therapy was later applied to the treatment of cancer. Such a genetic strategy seemed rational given the recognition that cancer typically develops in a multistep process involving alterations of a number of different genes as demonstrated in familial polyposis and colorectal cancer through the work of Vogelstein et al. Because of the numerous alterations that may result in the eventual development of cancer, there is no obvious single choice for a therapeutic gene. Although one may view this as an obstacle, it also allows for a variety of possible therapeutic interventions. This review focuses on the known genetic defects that occur in ovarian cancer, the gene therapy strategies suggested by such defects, and the approaches under current development for the treatment of this disease. As such, this work also describes some of the approved human gene therapy protocols. Finally, an overview of the problems and directions for future growth and research is presented.

Endothelial cell vehicles for delivery of cytotoxic genes as a gene therapy approach for carcinoma of the ovary.

Human umbilical vein endothelial cells (HUVECs) were evaluated for utility as a vector to achieve a bystander effect and killing of ovarian carcinoma cell lines. After demonstrating that HUVECs could be transduced with the reporter gene LacZ encoded by an adenoviral vector, appropriate cell killing of the AdCMVHSV-TK-transduced HUVECs was exhibited after treatment with 20 microM ganciclovir. Mixing experiments were then performed to determine whether the transduced HUVECs would demonstrate a bystander effect with the ovarian cancer cell lines. When 50% AdCMVHSV-TK-transduced HUVECs were mixed with untransduced ovarian cancer cells, > 70% of all cells were killed. Finally, s.c. and i.p. injections of herpes simplex-thymidine kinase-expressing HUVECs and SKOV3ip1 tumor cells were performed to evaluate the effects of HUVECs in in vivo models. These studies showed a decrease in tumor growth s.c. as well as a statistically significant survival prolongation (P < 0.05) in the i.p. model. These findings suggest that endothelial cells may be used as a vehicle for the delivery of cytotoxicity (bystander effect) in molecular chemotherapy.

Modulation of Bcl-2 protein levels by an intracellular anti-Bcl-2 single-chain antibody increases drug-induced cytotoxicity in the breast cancer cell line MCF-7.

Extensive experimental evidence suggests that Bcl-2 promotes cell survival by preventing the onset of apoptosis induced by a variety of stimuli. In addition, Bcl-2 expression has been correlated with resistance and poor response to chemotherapy in a number of cell types. Therefore, this protein represents a logical target for gene therapy strategies designed to achieve selective gene product ablation. In this study, we have developed an approach based upon intracellular expression of single-chain antibodies (sFvs) to achieve modulation of Bcl-2 protein levels in target cells. Using a transient expression system, we show that this intracellular anti-Bcl-2 sFv mediates specific reduction of Bcl-2 levels. This effect significantly enhances drug-mediated cytotoxicity in Bcl-2-overexpressing tumor cells, whereas transfection of the anti-Bcl-2 sFv did not affect the growth rate of the tumor cell lines. This method thus represents a novel and efficient way to selectively abrogate the activity of Bcl-2.

Intracellular expression of a single-chain antibody directed against human papillomavirus type 16 E7 oncoprotein achieves targeted antineoplastic effects.

Human papillomavirus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. Anti-HPV16 E7 intracellular single-chain antibodies (scFvs) were constructed to down-regulate HPV16 E7 oncoprotein in HPV DNA-containing cell lines. In these studies, we transfected anti-E7 scFvs into the HPV16-positive human cervical carcinoma cell lines CaSki and SiHa and tested them for their ability to inhibit cell proliferation and alter the level of HPV16 E7 oncoprotein. Our results showed that anti-HPV16 E7 scFvs inhibited cell proliferation by >85% in CaSki cells and by 95% in SiHa cells. E7 oncoprotein was down-regulated by anti-HPV16 E7 scFv, and its expression was inversely related to the amount of scFv transfected. However, there were no effects of transfecting scFvs alone in HPV-negative cell lines. These results imply that anti-HPV16 E7 scFvs only have specific anti-HPV16 E7 effects on cell proliferation and on the synthesis of virally encoded proteins in HPV-positive cell lines. Thus, transfection of HPV16 E7-positive tumors with antigen-specific scFvs may be a viable strategy for cervical cancer gene therapy.

Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector.

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The available vector systems have not allowed the simultaneous achievement of both goals. We have developed a chimeric viral vector system that incorporates favorable aspects of both adenoviral and retroviral vectors. Adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles are then able to stably transduce neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of a retroviral producer cell. The chimeric vectors may allow realization of the requisite goals for specific gene-therapy applications.

Conditionally replicative adenoviruses for cancer therapy.

The delineation of the genetic etiology of cancer makes gene therapy a rational approach for the molecular treatment of cancer. Many gene delivery systems have been developed, with viral vectors being the most effective. Underlying cancer gene therapy protocols is the recognition that quantitative tumor transduction cannot be achieved with the vector systems available at the present time. One way to overcome this problem could be to amplify the transduction efficiency through the use of vectors capable of replicating specifically in tumor cells. We are currently developing an adenoviral vector in which viral replication will be restricted to the target tumor cells by limiting the expression of viral genes essential for the virus replication only to the tumor cells of interest.

Adenoviral/retroviral vector chimeras: a novel strategy to achieve high-efficiency stable transduction in vivo.

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. Realization of both goals with available vector systems has so far not been achieved. As a novel approach to solve this problem, we have developed a chimeric viral vector system that exploits favorable aspects of both adenoviral and retroviral vectors. In this schema, adenoviral vectors induce target cells to function as transient retroviral producer cells in vivo. The progeny retroviral vector particles can then effectively achieve stable transduction of neighboring cells. In this system, the nonintegrative adenoviral vector is rendered functionally integrative via the intermediate generation of an induced retroviral producer cell. Such chimeric vectors may now allow realization of the requisite goals for specific gene therapy applications.

Mutagenesis of conserved residues of the adenovirus protease.

Based on the alignment of 12 adenovirus protease sequences, we have identified eleven conserved residues for mutagenesis. Eight of these, E5, D26, N44, E71, D77, D102, N144, and N170, are potential candidates for the third residue of the active site triad. N44, E71, N144, and N170 proved to be essential for enzyme activity. Glutamic acid 71 was proposed for the active site. Mutation of the three conserved cysteines suggested that C122 is the active nucleophile, C104 is the target for activation by peptide pVIc, and C126 is dispensable. Rescue of enzyme activity of the C104 G mutant by pVIc suggested that disulfide bond formation between the peptide and the protease may not be absolutely essential for stimulation of enzyme activity.