1796

Background: WWOX is a candidate tumor suppressor gene that maps to 16q23.3-24.1 and encodes a 9-exon, 2.2kb transcript. Homozygous deletions within WWOX have been reported in a variety of cancer cell lines and in human primary tumor material. High frequency of allelic loss involving WWOX has been shown in ovarian, esophageal and non-small cell lung cancers. Aberrant, exon-skipped isoforms of the gene have been identified in cell lines and tumor tissue. Purpose: To identify a cell line system in which the WWOX pathway was intact (or reconstitutable) to facilitate the identification of an in vitro phenotype and the further investigation of the role of WWOX as a tumor suppressor. Materials and Methods: The HCT116 colorectal and PEO1 and A2780 ovarian cancer cell lines were transfected with sense and antisense constructs to the WWOX open reading frame and the level of expression of WWOX in the resultant transfectants was quantified using real-time PCR. WWOX protein expression was also checked in the PEO1 cell line by Western Blot. Phenotypic analyses were initially conducted on the HCT116 antisense clones and the PEO1 sense clones on the basis of the WWOX isoform expression of the respective parent lines (HCT 116 cells express full length WWOX, the Δ6-8 WWOX transcript as well as lower amounts of other exon-skipped forms of the gene whereas the PEO1 cell line is homozygously deleted for exons 4 to 8 and only expresses small amounts of a Δ4-8 transcript). Results: No effect on proliferation in vitro or tumorigenicity in nude mice was detected in the HCT116 antisense clones. In the PEO1 sense transfectants however, there was a complete abolition of tumorigenicity in nude mice in all clones. The PEO1 parent line expressed no full length WWOX transcript and produced tumors in nude mice. The abolition of tumorigenicity in the WWOX transfected clones suggested the presence of a reconstituted WWOX pathway in the transfected PEO1 cell line system so an in vitro phenotype for the gene was sought. In vitro phenotypic analysis revealed no difference in growth (either in plastic or soft agar), clonogenicity (in presence or absence of cytotoxic drugs), cell aggregation or cell invasion for the WWOX transfectants. We did however show decreased cell migration towards fibronectin and laminin (but not collagen IV). Preliminary data also suggested that replacement of WWOX resulted in decreased adherence to matrix components. A strong correlation between WWOX mRNA and protein levels in the PEO1 transfectants was confirmed (R2=0.995). Conclusion: Reconstitution of WWOX expression in the PEO1 ovarian cancer cell line results in abolition of tumorigenicity in nude mice and in decreased cell migration towards fibronectin and laminin. These findings support the hypothesis that WWOX is a suppressor of ovarian tumorigenesis and provide a system for further dissection of the phenotype of the gene.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]