A strong body of work has implicated the cell membrane glycolipid-linked urokinase receptor (u-PAR) in promoting tumor growth and progression. u-PAR promotes these effects in two distinct ways. First, it interacts with a5?1 integrin thereby increasing the ratio of activated ERK/p38 yielding an increased growth fraction. Second, the u-PAR binds the serine protease urokinase thus increasing plasmin formation and extracellular matrix degradation, tumor cell migration and invasion. However, the regulation of u-PAR expression is poorly understood. We have made the intriguing observation that in some colon cancer cells, clonal populations oscillate in u-PAR display between high and low cell surface density with reduced tumorigenecity segregating with the latter. Interestingly, the altered u-PAR cell surface display is post-translational in nature reflecting a shift in u-PAR trafficking.
In Specific Aim #1 we will determine the prevalence of u-PAR display plasticity in colon cancer and whether altered tumorigenecity mirrors this oscillation in cell surface u-PAR density. Moreover, we will determine if a shift in the ratio of activated ERKs/p38 downstream of oscillating u-PAR display dictates tumorigenic potential in these metastable cell populations. Finally, we will determine whether u-PAR secretion or shedding is the mechanism responsible for u-PAR display plasticity. If the latter, we will employ expression profiling and siRNA/shRNA experiments to identify protein(s) in the GPI anchoring or shedding machineries responsible for modulated u-PAR display. To date, there are few known biological regulators of u-PAR expression.
In Specific Aim # 2, we will employ an unbiased expression cloning strategy to identify novel regulators of u-PAR expression. Candidate regulators will be validated by over-expression/knockdown experiments in tissue culture and by employing mice gene-trapped for the regulator to determine if u-PAR expression is modulated in tissues that in wt animals co-express the candidate regulator and u-PAR. Finally, we will correlate, by immunohistochemistry, the amounts of regulator and u-PAR in resected colon cancer sections to accrue further evidence for a role of the regulator in driving u-PAR expression in this malignancy. Previously, we described 1469 bp of upstream sequence as regulatory for u-PAR expression. However, based on our transfection experiments with reporter constructs and studies with transgenic mice harboring a LacZ reporter driven by this upstream sequence, we have concluded that this sequence while necessary for is, nevertheless, insufficient to faithfully mirror endogenous gene expression. We describe herein a novel enhancer residing in a chromatinized region of intron 1 (+665/+2068) of the u-PAR gene, that contributes to optimal expression in colon cancer. These findings lead to Specific Aim # 3 where using chromatin immunoprecipitation assays and dominant negative expression technology we will identify enhancer-bound co-regulator(s) controlling u-PAR expression. Additionally, we will determine whether this intronic enhancer confers tissue-specific (placenta, wounded skin) u-PAR expression in mice.
| Ishiguro, T; Avila, H; Lin, S-Y et al. (2010) Gene trapping identifies chloride channel 4 as a novel inducer of colon cancer cell migration, invasion and metastases. Br J Cancer 102:774-82 |
