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The receptor for macrophage colony-stimulating factor 1 receptor (CSF1R) is a product of the proto-oncogene c-fms and a member of the class III transmembrane tyrosine kinase receptor family. Earlier, we described increased mRNA expression of CSF1R in human telomerase reverse transcriptase (hTERT) immortalized human ovarian surface epithelial (IOSE) cell lines derived from a single donor. Here, we further describe that CSF1R is upregulated at both the mRNA and protein level in hTERT immortalized human normal OSE cells from two different donors and in hTERT immortalized human pancreatic ductal epithelial cells. CSF1R was not upregulated in hTERT immortalized epithelial clones that subsequently underwent senescence or in immortalized fibroblasts. Upon stimulation by the CSF1R ligand CSF1, the immortalized epithelial cell lines showed rapid internalization of CSF1R with concomitant down-modulation and colocalization of phosphorylated NFkappaBp65 with hTERT protein, hTERT translocation into the nucleus and the binding of c-Myc to the hTERT promoter region. Reducing the expression of CSF1R using short hairpin interfering RNA abolished these effects and also decreased cell survival and the number of population doublings under suboptimal culture conditions. The telomerase inhibitor GRN163L confirmed a role for telomerase in the cleavage of the intracellular domain of CSF1R. On the basis of these findings, we suggest that CSF1R may be a critical factor facilitating hTERT immortalization of epithelial cells.

Constitutive production of inflammatory cytokines is a characteristic of many human malignant cell lines; however, the in vitro and in vivo interdependence of these cytokines, and their significance to the human cancer microenvironment, are both poorly understood. Here, we describe for the first time how three key cytokine/chemokine mediators of cancer-related inflammation, TNF, CXCL12, and interleukin 6, are involved in an autocrine cytokine network, the “TNF network,” in human ovarian cancer. We show that this network has paracrine actions on angiogenesis, infiltration of myeloid cells, and NOTCH signaling in both murine xenografts and human ovarian tumor biopsies. Neutralizing antibodies or siRNA to individual members of this TNF network reduced angiogenesis, myeloid cell infiltration, and experimental peritoneal ovarian tumor growth. The dependency of network genes on TNF was shown by their downregulation in tumor cells from patients with advanced ovarian cancer following the infusion of anti-TNF antibodies. Together, the findings define a network of inflammatory cytokine interactions that are crucial to tumor growth and validate this network as a key therapeutic target in ovarian cancer. Cancer Res; 72(1); 66–75. ©2011 AACR.

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.

The oncolytic adenovirus dl 922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl 922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl 1520 (Onyx-015). We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl 922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl 922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1) to 0.03 pfu/cell (TOV21G). Cells sensitive to dl 922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl 922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.

Introduction. Oncolytic adenoviruses show promise as a cancer treatment, by replicating selectively within malignant cells. Viral replication requires multiple cell cycle checkpoints to be overcome. Ovarian cancer sensitivity to viral cytotoxicity varies greatly, even between cells with similar infectivity and intracellular virion production. Viruses also generate acute inflammatory responses with production of cytokines, especially TNF-α. We investigated the role of cellular DNA damage in efficacy of adenovirus dl922-947 in ovarian cancer and also whether inhibition of TNF-α augments activity. Methods. Host DNA damage and repair pathways were assessed in ovarian cancer lines, TOV21G, IGROV1 and A2780CP, which cover three-log range of virus sensitivity. The ATR-chk1 pathway was inhibited using siRNA and UCN-01 treatment. In addition, inflammatory cytokine responses to dl922-947 were assessed in vitro and in xenograft models. TNF-α was inhibited using shRNA and monoclonal antibodies. Results. Oncolytic adenovirus cytotoxicity is associated with extensive genomic DNA damage. Following infection, sensitive, but not resistant, ovarian cancer cells activate cdc25a and over-replicate genomic DNA, creating extensive double-strand breaks. Inhibition of ATR-chk1 in resistant cells augments cdc25a activity and increases genomic DNA over-replication. This enhances cytotoxicity without increasing E1A expression or viral replication. In addition, dl922-947 induces transcription of TNF-α, IL-6 and IL-8 in ovarian cancer cells and xenograft-bearing mice prior to any viral gene expression and host NF-κB activation. Both RNAi-mediated TNF-α knockdown and anti-TNF-α monoclonal antibody treatment increase virus cytotoxicity in vitro and in IGROV1 intraperitoneal xenografts. The increased efficacy results from suppression of cIAP1 transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. Conclusions. A phase I trial of dl922-947 in women with relapsed ovarian cancer is planned for 2011. Host cell DNA damage is a key determinant of oncolytic adenoviral activity, whilst TNF-α acts as a survival factor in infected cells. Inhibiting both repair of DNA damage and TNF-α could improve anti-tumour activity in future clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 591.