Abstract 432: Expression of PITX2 is correlated with invasiveness in breast cancer cells and its downregulation reduces invasive potential

Objective We have previously found that PITX2 expression in the bone marrow (BM) of women newly diagnosed with breast cancer correlates with the development of distant disease. PITX2 is not expressed in the BM of normal volunteers, and thus the source of PITX2 is likely to be disseminated tumor cells which lodge in the bone marrow. PITX2 plays a well-defined role in development. This study was conducted to understand the role of expression of PITX2 in the breast cancer phenotype. Experimental procedures PITX2 has three different transcript variants. The RNA expression level of each transcript variant was determined by qRT-PCR using specific primers designed for each isoforms in a variety of breast cancer cell lines. PITX2 was stably silenced using shRNA lentiviral constructs against all isoforms of PITX2 (Sigma, St. Louis) and selecting with puromycin. Individual clones were selected and tested for PITX2 expression. Those cells with near total knockdown of the gene were used for matrigel invasion assays using manufacturer's protocols. Briefly, about 3 × 104 cells were suspended in growth factor free media and added to each well of invasion chamber and complete growth medium was added to the lower chamber as chemoattractant. The number of cells which invaded was counted at 24h and 48h after seeding. Control cells included empty vector, a non-targeting sequence, and shRNA against the unrelated gene, Beta 2- Microglobulin. The number of invasive cells were counted from five different fields from each well and tallied. Percentage invasion was calculated as the number of cells passed through the Matrigel to the total number of cells before harvest. All experiments were repeated three times. Results PITX2 isoforms A and B were found to be expressed in the invasive cell lines MDA MB 231 and C1A1 while none of the isoforms were expressed in non-invasive cell lines MCF7 and MCF10A. In matrigel invasion assay, the knockdown of PITX2 substantially reduced invasiveness compared to the controls. There was a 63.8 % reduction in invasion in PITX2 deficient cells at 24 hrs and 72.6 % reduction at 48 hrs compared to the parental MDA MB 231 cells. ConclusionPITX2 is expressed in breast cancer cell lines which have the ability invade and is not expressed in cell lines which cannot invade. Silencing of PITX2 in breast cancer cell lines abrogates the ability of breast cancer cells to invade. These findings combined with our clinic data suggest that PITX2 plays an important rol