Prognosis-driven drug discovery for human colon cancer

A59 Treatment of colon cancer patients would benefit from the development of an accurate prognostic test that could also serve to guide therapeutic decisions. In an effort to meet this goal, we sought to i) develop a prognostic quantitative real-time RT-PCR assay using formalin fixed paraffin-embedded primary tumors, and ii), develop an assay that could be used for high throughput screening of drugs that would either abolish or restore functional activity of a viable target identified in the prognostic assay. To achieve these two objectives, we first measured the expression levels of 14 cancer-associated genes in Stage II colon cancer patients who developed disease recurrence within two years (n=9), or those who did not develop disease recurrence (n=12, mean follow-up time = 6.3 yrs). We observed that the expression ratio of (EpCAM1+Map7)/E-cadherin accurately predicted clinical outcome (Hazard ratio = 12; P=0.001 log-ranked test), such that good patient survival was associated with high expression of E-cadherin, a gene that has been shown to be downregulated by Zeb1 during colon cancer progression. Interestingly, we discovered that whereas the expression of E-cadherin was inversely correlated with Zeb1 (p=1.4E-7) in the CGAP NCI60 database, the expression of EpCAM1 was inversely correlated with Zeb2 (p=1.9E-10). We further discovered that the promoter region of EpCAM1 contained a 16 nucleotide match to the region including and surrounding the Zeb1 response element of the p73 gene (p=8.7E-7). Based on these findings, we concluded that E-cadherin represented the most viable therapeutic target of the three genes identified in the prognostic assay. To enable screening for drugs that might restore E-cadherin function, we developed a 20 microliter adhesion assay using an uncoated polystyrene surface in which it was observed that following 7 hours after cell trypsinization, the level of adhesion of highly metastatic SW620 colon cancer cell line was 15-fold (+/-4) lower compared to poorly metastatic HCT116, a cell line which we determined contains 100- and 8-fold higher levels of E-cadherin mRNA and protein, respectively, compared to SW620. This adhesion assay may allow for the identification of novel compounds that inhibit the epithelial-mesenchymal transition required for spread of metastatic disease.