Epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses - PubMed (original) (raw)

. 2009 Jun 15;69(12):5115-25.

doi: 10.1158/0008-5472.CAN-09-0645. Epub 2009 Jun 2.

Pavel Sova, Ying Liu, Zong Yi Li, Sebastian Tuve, David Pritchard, Paul Brinkkoetter, Thomas Möller, Oliver Wildner, Sari Pesonen, Akseli Hemminki, Nicole Urban, Charles Drescher, André Lieber

Affiliations

Epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses

Robert Strauss et al. Cancer Res. 2009.

Abstract

We studied the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie-adenovirus receptor or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, thus far little-explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14) that use receptor(s) other than coxsackie-adenovirus receptor and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed several interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (epithelial/mesenchymal cells). These epithelial/mesenchymal cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.

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Figures

Fig.1

Fig.1. Analysis of clonal ovarian cancer cultures that were either resistant or susceptible to lysis by Ad5/35.IR-E1A/TRAIL

A) Cytolysis caused by Ad5/35.IR-E1A/TRAIL in clonal cultures derived from biopsy ovc316. Clonal cultures indicated in black were subjected to DNA expression array analysis. Standard deviation was less than 10% of the average for all samples. Infection at MOIs 10 and 200 pfu/cell resulted in a similar distribution. B) Focal adhesion, tight junction (TJ) and adherens junction (AJ) pathways. Genes that were found upregulated in arrays are marked red. Down-regulated genes are marked green. TJ proteins, include claudins and occludin. AJ proteins include E-cadherin. The cytoplasmic domain of E-cadherin interacts with ß-catenin and p120-catenin. Claudins and occludin interact with ZO-1, and subsequently with F-actin via cingulin. Caldesmon inhibits Arp2/3-mediated actin polymerization. Phosphoinositide 3-kinase (PI3K) and Focal adhesion kinase (FAK) are involved in the regulation of Rho GTPases downstream of integrin signaling. Rho kinase (ROCK) is activated by RhoA. Vinculin and α-actin crosslink the cytoskeleton to focal adhesion spots. Vimentin is the major intermediate filament (IF) protein of mesenchymal cells that is involved in regulation of attachment, migration, and cell signaling. Palladin functions as a scaffold that regulates actin organization. Profilin is involved in turnover of the actin filament network. C) Flow cytometry analysis of resistant and susceptible clones. Two representative clones are shown. D) Immunofluorescence analysis of epithelial marker proteins (upper panels) and mesenchymal marker proteins (lower panels) on resistant and susceptible clones. Representative clones are shown.

Fig.2

Fig.2. Analysis of primary ovarian cancer culture ovc316 for epithelial and mesenchymal markers after infection with Ad5/35.IR-E1A/TRAIL

A) Primary cultures (passage 10) were infected with Ad5/35.IR-E1A/TRAIL at an MOI of 100 pfu/cell and analyzed at day 2, 4, and 8 after infection for immunofluorescence of as indicated. Uninfected cells were used as a control. B) Flow cytometry of infected cells. Representative samples are shown. Notably, resistant cells that showed cytoplasmic E-cadherin staining in immunofluorescence studies scored negative by flow cytometry analysis for surface E-cadherin.

Fig.3

Fig.3. Analysis of adenovirus receptors

A) Ad infection of R/E and S/M cells. Left panel: Attachment of 3H-labeled particles to cells. Middle and right panels: Virus uptake and genome replication. The amount of viral genomes in cells was measured 3 and 72 hours after infection with Ad vectors at an MOI of 100 pfu/cell by qPCR. B) Flow cytometry analysis of surface CD46 and αV integrins on R/E (resistant) and S/M (susceptible) clones. C) Confocal microscopy analysis of CD46, αV integrin, and claudin 7 on cells that were incubated with Cy3-labeled Ad5/35.IR-E1A/TRAIL (4,000 vp/cell) on ice for 30 min (“attachment”) or incubated with virus for 2 hours at 37°C (“internalization”). D) Infection of cells with Ad5/35.GFP from the apical and basal side. Clonal cultures were seeded using tissue culture inserts in 48 well plates and virus was added to medium on top or below cells at an MOI of 10 or 100 pfu/cell. GFP expression was analyzed 48 hours later by flow cytometry.

Fig.4

Fig.4. Analysis of ovc316 xenografts

A) Analysis of tumor sections. Upper panel: Expression of E-cadherin (green) and laminin (red) Middle panel: Co-localization of CD46 (green) or αV integrins (green) with the tight junction protein claudin 7 (red). Lower panel: Viral hexon and E-cadherin expression at day 8 post injection of 2×109 pfu of Ad5/35.IR-E1A/TRAIL; In vivo GFP expression after intratumoral injection of Ad5/35.GFP. In vivo βGal expression after intravenous injection of Ad5/35. βGal. B) Flow cytometry of cell suspensions and cultured ovc316 cells at passage 1 and 20. C) Western blot for key members of pathways that regulate tight junction reorganization and EMT. D) Effect of inhibitors {exoenzyme C3 from Clostridium botulinum (inhibitor of Rho A, B and C GTPases), H-1152 (Rho-kinase inhibitor), Clostridium difficile toxin B (inhibitor for Rho, Rac, and Cdc42), Wortmannin (inhibitor of PI3K)} on viability of Ad5/35.IR-E1A/TRAIL infected R/E and S/M cells. Cell viability was measured at day 4 after infection at an MOI of 100 pfu/cell. Significance was compared to infected, Mock treated cells. ***p<0.001, **p<0.01, *p<0.05

Fig.5

Fig.5. Correlation between epithelial and mesenchymal phenotype of liver metastases and Ad5/35 vectors transduction after intravenous injection

Human tumor cells (HeLa, SK-Hep1, SAOS, or HT-29) were injected into the portal vein of immunodeficient mice. After liver metastases formed, mice received a tail vein injection of 2 × 109 pfu of Ad5/35 vectors expressing either GFP or βGal. Tumor-bearing livers were analyzed 3 days later for claudin 7 and transgene expression. HT-29 tumor bearing liver sections were also stained for CD31. For SK-Hep1 tumors, viral particles were visualized with an anti-hexon-FITC antibody at 2 hours post-injection.

Fig.6

Fig.6. Analysis of infectivity of ovarian cancer cells by different Ad serotypes

A) Immunofluorescence analyses on R/E cells. Left upper panel: Expression of HSPGs (green) and claudin 7 (red) on R/E cells. Left lower panel: Cy3-Ad3 and Cy3-Ad35 attachment in R/E cells (right panel). Right panel: Effect of Ad 3, 5, 7, 11, 14, and 35 infection (MOI 100pfu/cell) on cell morphology and E-cadherin (green) expression in R/E cells. Viral replication was visualized by staining for hexon (red). The analysis was done at day 4 p.i. B) Flow cytometry analyses of Ad infected cells (same conditions as in B). C and D) In vivo transduction of wAd3 and wtAd35. A total of 2×109 pfu of Ad3 and Ad35 was intratumorally injected into subcutaneous ovc316 tumors. C) Transduction was quantified by qRT-PCR for hexon mRNA using pan-serotype hexon primers that can detect both Ad3 and Ad35 hexon mRNA (23). N=5. D) Ovc316 tumor volume after mock injection or intratumoral injection of 2×109 pfu of wtAd3 or wtAd35, N=5.

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