Dysregulation of mitotic spindle checkpoint control in hepatocellular carcinomas

Abstract

Chromosomal instability (CIN) refers to high rates of chromosome gains and losses and is a major cause of genomic instability. Recurrent chromosome alterations have been commonly found in hepatocellular carcinoma (HCC), which is a prevalent cancer worldwide and in Southeast Asia and Hong Kong. These frequent gains and losses of chromosomes suggest that CIN contributes to hepatocarcinogenesis. One major mechanism for CIN is through the loss of mitotic checkpoint. In this study, we evaluated mitotic spindle checkpoint competence in HCC cell lines, using nocodazole and colcemid, which inhibit spindle assembly. Six of the eleven HCC cell lines tested showed a defect in mitotic competency. In addition, these six HCC cell lines also showed a reduction in the expression of a major mitotic checkpoint protein, called Mitotic Arrest Deficient 2 (MAD2). This association of mitotic spindle checkpoint incompetence and the downregulation of MAD2 protein in these HCC cells suggest that deficiency of the MAD2 protein may play a role in mitotic spindle checkpoint defect in HCC. In addition to MAD2, we have also examined the expression of MAD1, the binding partner of MAD2, which also plays an important role in the progression of mitosis. During the evaluation of MAD1 expression, we have identified a novel splicing variant of MAD1, which we named MAD1β. This isoform was detected in all HCC cell lines tested and in most of the human HCC samples and their corresponding nontumorous livers. Upon sequencing, we found that MAD1β had no exon 4, which was likely a result of different splicing. In addition, with confocal microscopy, overexpression of MAD1β in HCC cell lines resulted in a different subcellular localization (cytoplasmic) as compared with that of MAD1 (nuclear). These results suggest that exon 4 may contain a nuclear localization signal which can regulate the localization of MAD1 protein in cells. Overall, our results suggest that reduced MAD2 expression may contribute to mitotic checkpoint defect in HCC. In addition, further investigation into this newly identified MAD1 isoform, MAD1β, is warranted.