DNA aneuploidy or chromosomal instability is commonly observed in major types of human cancers, and one of the causes for chromosomal instability is defective mitotic checkpoint signaling in cells. Loss of mitotic checkpoint contributes to chromosomal instability, leading to carcinogenesis. The mitotic checkpoint monitors the status of kinetochore-microtubule attachment and delays the onset of anaphase until all kinetochores have been attached with microtubules. Mitotic arrest deficient 1 (MAD1) is key component in mitotic checkpoint signaling. We previously identified a novel isoform of MAD1, which we named MAD1β (Sze et al. Cancer Res 2008;68:9194-9201). In this study, using yeast-two hybrid screening, we have identified a novel MAD1 interacting partner, proline-rich acidic protein 1 (PRAP1). The function of PRAP1 is largely unknown. Hence, we investigated its role in mitotic checkpoint signaling in hepatocellular carcinoma (HCC). We demonstrated the physical interaction between PRAP1, MAD1 and MAD1β in yeast-two hybrid screening and with co-immunoprecipitation assay. Both MAD1 and MAD1β proteins were co-immunoprecipitated with PRAP1. Moreover, we stably expressed PRAP1 in two HCC cell lines, BEL-7402 and SMMC-7721, which have competent mitotic checkpoints. We observed that stable expression of PRAP1 induced impairment in mitotic checkpoint (P < 0.01). After treatment with either colcemid or nocodazole, the PRAP1-stably expressing BEL-7402 and SMMC-7721 cells showed significantly lower mitotic indices as compared with the corresponding vector control cells. Ectopic expression of PRAP1 also resulted in chromosome bridge formation (P< 0.01) and aberrant chromosome numbers (P < 0.001) in these cell lines. All these cellular changes contributed to chromosomal instability. Interestingly, ectopic expression PRAP1 led to significant underexpression of MAD1 but not MAD1β in HCC cells. Taken altogether, our data indicate that PRAP1 is an interacting partner of MAD1 and may have functions of degrading MAD1 and in turn suppressing mitotic checkpoint signaling in HCC.

(This study was partly funded by a Hong Kong Research Grants Council GRF grant [HKU 772608])

Citation Format: Karen MF Sze, Glanice K. Chu, Queenie HY Mak, Irene OL Ng. Proline-rich acidic protein 1 (PRAP1), a novel mitotic arrest deficient 1 (MAD1) interacting partner, suppresses mitotic checkpoint signaling in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2013-572