Correlation of PITX2 gene expression and invasive potential of breast cancer cells.

Abstract

10646 Background: We have found that expression of PITX2, a gene normally involved in development, in the bone marrow (BM) of women with newly diagnosed breast cancer correlates with the early development of distant metastatic disease. PITX2 is not expressed in the BM of healthy volunteers, suggesting that the source of PITX2 expression is disseminated tumor cells. This study was conducted to understand PITX2 expression as it relates to the metastatic breast cancer phenotype. Methods: PITX2 has three different transcript variants. The expression of each transcript variant was quantified by qRT-PCR. PITX2 expression in the MDAMB231 cell line was stably silenced using shRNA lentiviral constructs against all isoforms of PITX2 (Sigma, St. Louis) and selecting with puromycin. Cells with total knockdown of the gene were used for matrigel invasion assays. Briefly, 3 × 104 cells were suspended in growth factor free media and added to each well of an invasion chamber. Complete growth medium was added to the lower chamber as chemoattractant. The number of invasive cells was counted at 24h and 48h after seeding. Control cells included empty vector, a non-targeting sequence, and shRNA against the unrelated gene, Beta2-Microglobulin. Percentage invasion was calculated as the number of cells passing through the Matrigel to the total number of cells present before harvest. All experiments were repeated three times. Chi-square was used for analysis. Results: PITX2 isoform A was expressed in the invasive cell lines MDA MB 231 and C1A1 while isoform C was expressed in non-invasive cell lines MCF7 and MCF10A. In the matrigel invasion assay, knockdown of PITX2 in MDA MB231 reduced invasiveness 3-fold compared to the controls at 24 and 48 hours (p < 0.0001). Conclusions: The level of PITX2 expression in several human breast cancer cell lines is correlated with their invasive potential. Down-regulating PITX2 transcript expression abrogates the invasive phenotype. These functional results corroborate our previous clinical findings that PITX2 expression is associated with disseminated tumor cells and the rapid development of disease. PITX2 in disseminated tumor cells may serve as a new therapeutic target in preventing the development of metastatic disease. No significant financial relationships to disclose.