Occurrence of Aurora A positive multipolar mitoses in distinct molecular classes of colorectal carcinomas and effect of Aurora A inhibition

Abstract

Aurora A "over-"expression may induce supernumerary centrosomes, respective multipolar mitoses, and aneuploidy. Here, we examined Aurora A positive multipolar mitoses in aneuploid, microsatellite-stable (MSS, "CIN-type") versus near-diploid, microsatellite-instable (MSI, "MIN-type") colorectal carcinomas (CRC) and CRC cell lines as well as the effect of Aurora A inhibition in CRC cell lines. In situ, three-dimensional immunofluorescence (3D-IF) revealed Aurora A positive multipolar mitoses in both CIN- (n = 8) and MIN- (n = 10) type primary CRCs with similar frequencies (CIN: 27 ± 14%; MIN: 34 ± 14%, P = 0.224). In vitro, Aurora A positive multipolar mitoses were detected in asynchronized or thymidine synchronized CIN-type (HT29, CaCo-2), but not MIN-type (HCT116, DLD-1) CRC cells. Nocodazole treatment arrested mitotic cells with multiple centrosomal Aurora A signals in CIN- and MIN-type CRC cells, albeit to a lower extent in CaCo-2 cells. This was associated with concomitant activation of Aurora A (T288 phosphorylation) and Polo-like kinase 1 (PLK-1, T210 phosphorylation). Aurora A inhibition by siRNA resulted in increased apoptosis (>50%) in all cell lines, but did not abolish PLK-1 expression. Double 3D-IF revealed that Aurora A siRNA treated, still viable CIN-type (HT29, CaCo-2) CRC cells were Aurora A negative and mostly in prophase/(pro)metaphase with maintained phosphorylated PLK-1 T210 expression. Aurora A positive multipolar mitoses occur in both aneuploid, CIN- and near-diploid MIN-type CRCs. This appears to be largely independent of Aurora A expression alone. Although Aurora A inhibition causes apoptosis in both CIN- and MIN-type CRC cells, remaining PLK-1 activation by other factors may affect therapeutic Aurora inhibition.