Abstract
The frequent loss of heterozygosity of chromosome (Chr) 17 in epithelial ovarian cancer (EOC), particularly high-grade ovarian serous carcinomas (HGOSCs), has been attributed to the disruption of known tumour suppressor genes, such as TP53 (17p13), as well as other genes on this chromosome that alone or in combination have a role in EOC. In a transcriptome analysis of Chr17 genes, we observed significant underexpression of the chemokine CCL2 (17q12) in a small set of HGOSC samples relative to normal ovarian surface epithelial cells and a significant upregulation of CCL2 in the TP53-mutated OV-90 EOC cell line rendered non-tumourigenic as a consequence of genetic manipulation. Here, we report that overexpressing CCL2 in OV-90 resulted in latency of tumour formation at intraperitoneal (i.p.) but not subcutaneous sites in a mouse xenograft model. Overexpressing CCL2 affected cell morphology and exerted modest, but not significant effects on cell viability, colony formation and cell migration. We report significant underexpression of CCL2 by transcriptome analysis (P=0.015) and by immunohistochemistry in 77% of HGOSC samples (n=65). Absent or a very low level of protein expression by immunohistochemistry was also observed in 71% of additional HGOSC samples (n=122). However, CCL2 protein expression did not significantly correlate with overall or disease-free survival. The epithelial cells of normal fallopian tubes, a purported origin of HGOSC, exhibited expression of CCL2 protein by immunohistochemistry. Our results affirm that CCL2 underexpression is a significant feature of HGOSC samples, and that CCL2 overexpression in an EOC cell line model affects tumourigenic potential in the i.p. setting.
Highlights
A well-documented feature of epithelial ovarian cancers (EOCs) is the high frequency of loss of heterozygosity of a chromosome (Chr) 17 contig.[1,2,3,4,5] This observation along with complementation studies showing a reduction in tumourigenicity of an EOC cell line harbouring a transferred normal Chr[17,6] have suggested thatChr[17] harbours tumour suppressor genes (TSGs)
In a recent transcriptome analysis, we described Chr[17] genes dysregulated in high-grade ovarian serous carcinomas (HGOSCs) as compared with normal ovarian surface epithelial cells (NOSEs),[19] and showed that the chemokine CCL2 (17q12) was one of the genes significantly underexpressed in HGOSCs.[19]
Using severe combined immunodeficiency mice, the CCL2-expressing clones were tumourigenic at the s.c. site, where there were no significant differences in the time-to-kill, final mean tumour volume or mean tumour growth rate as compared with OV-90 (Figure 3, Supplementary Table S2)
Summary
A well-documented feature of epithelial ovarian cancers (EOCs) is the high frequency of loss of heterozygosity of a chromosome (Chr) 17 contig.[1,2,3,4,5] This observation along with complementation studies showing a reduction in tumourigenicity of an EOC cell line harbouring a transferred normal Chr[17,6] have suggested that. Two OV-90 clones, OV-90:CCL223 and OV90:CCL249, were selected from the 47 clones obtained in cell transfection assays with the CCL2-expressing construct They were selected for further analysis based on the high level of CCL2 protein and gene expression as compared with RH-6 and the parental OV-90 cell line (Figures 1a and b, Supplementary Table S1). OV-90 cells are able to form tumours when injected at both subcutaneous (s.c.) and intraperitoneal (i.p.) sites in immunocompromised mice, where there is an accumulation of ascites at the i.p. site.[31] Using severe combined immunodeficiency mice, the CCL2-expressing clones were tumourigenic at the s.c. site, where there were no significant differences in the time-to-kill, final mean tumour volume or mean tumour growth rate as compared with OV-90 (Figure 3, Supplementary Table S2).
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