In this study, we showed for the first time that the Cul4A gene is amplified in human mesothelioma cell lines. Consistent with gene amplification, overexpression of Cul4A protein was observed in mesothelioma cell lines and MPM tissues. Further knockdown of Cul4A by shRNA in mesothelioma cells also inhibited cells growth. In the contrary, overexpression of ectopic Cul4A in mesothelioma cells promoted cells growth. Thus, our results indicate that amplification of Cul4A gene may be an important oncogenic event in mesothelioma development.
We also observed that down-regulation of Cul4A with shRNA causes cell cycle arrest and growth inhibition through up-regulation of p21 and p27 proteins in a p53-independent manner in mesothelioma cells. Several lines of evidence support this hypothesis. First, down-regulation of Cul4A with shRNA increased the levels of p21 and p27 in the p14ARF-null mesothelioma cell lines (H290 and H28) we studied, whereas p53 protein levels were unchanged in these cell lines. Second, down-regulation of Cul4A in the p53-null HCT-116 cells increased p21 and p27 protein levels. Third, a very recent report provided the evidence of direct degradation of p21 through a Cul4-DDB1Cdt2
], suggesting that up-regulation of p21 may be due to decreased proteolysis of p21 after Cul4A knockdown without participation of p53. Previous work has also shown that Cul4A-DDB1 associates with SCFSKP2
, and participates in p27 degradation between G1/S and S phase [18
]. In short, these studies suggest that Cul4A can regulate p21 and p27 protein levels in a p53-independent manner, probably through direct ubiquitination and proteolysis of p21 and p27 proteins. Furthermore, the results of our colony formation assay provide additional evidence that the p14ARF-null mesothelioma cell lines are more dependent on Cul4A regulation than is the mesothelioma cell line (MS-1) with an intact p14ARF-p53 pathway. More dramatic inhibition of colony formation was noticed in the two p14ARF-null cell lines: H290 and H28, whereas only modest inhibition was noticed in the MS-1 cell line. MS-1 cells, which still retain expression of p14ARF [19
], showed increased p53 and p21 after knockdown of Cul4A, indicating that up-regulation of p21 may occur in both a p53-dependent and p53-independent manner after knockdown of Cul4A. Taken together, our findings suggest that Cul4A shRNA may be more effective in cancer cells that lack the intact p14ARF-p53 pathway. Since most human cancer cells lack the intact pathway, the Cul4A complex may be a potential interesting target for cancer therapy.
In this study, up-regulation of p21 and p27 proteins after Cul4A knockdown and down-regulation of p21 and p27 proteins after expression of ectopic Cul4A were observed. Our data suggests that both p21 and p27 proteins are at least partially regulated by Cul4A. Both p21 and p27 are important regulators of cell cycle and cell growth. p21 is a cyclin-dependent kinase inhibitor encoded by the growth inhibitory gene p21waf1/cip1
, and exerts its roles in cell-cycle arrest. Deficiency of p21 protein is associated with abrogation of cells that undergo G1 arrest after DNA damage in p21 knockout mice [20
] and colon cancer cells [21
]. Overexpression of p21 in hamster BHK21 cells causes cell cycle arrest in G1 phase, and reduces cell growth and DNA synthesis [22
]. In addition to p21 overexpression, we noted up-regulation of p27 after Cul4A knockdown. p27 is also a cyclin-dependent kinase inhibitor, and its expression inversely correlates with poor patient prognosis in a large variety of cancers [23
]. In mouse models, p27 has been noted to be a haplo-insufficient tumour suppressor [26
]. It is tightly regulated in G0, G1 and S phases, and is elevated in G0 phase or growth factor-depleted cells. In contrast, degradation of p27 has been noted as cells are released from G1 and reaches the lowest levels in the late G1 and S phases [27
]. Furthermore, over-expression of p27 was found to prevent activation of cyclin-dependent kinases and entry into the S phase of the cell cycle [28
]. In replicating cells, the well-known p27 regulatory pathway involves proteasomal degradation of p27, which requires polyubiquitination by the SCFskp2
-E3 ligase in late G1 and early S phase [29
]. Previous work has shown that inactivation of the Cul4A ubiquitin E3 ligases pathway by siRNA knockdown of Cul4A is associated with p27 stabilization and p27-dependent G1 cell cycle arrest in human cancer cells [7
]. However, the association of Cul4A knockdown and p21 up-regulation in human cancer cells has not been elucidated.
Our study showed that down-regulation of Cul4A causes G0/G1 cell cycle arrest and then growth inhibition in mesothelioma cells, and that siRNA knockdown of p21 and/or p27 restored G0/G1 cell cycle arrest in Cul4A knockdown mesothelioma cells. Interestingly, knockdown of p21 and/or p27 at least partially reversed in Cul4A stably knockdown mesothelioma cells. Importantly, up-regulation of p21 seems also to have effects on G0/G1 arrest in Cul4A knockdown mesothelioma cells, because restoration effects of G0/G1 arrest appear earlier and stronger after siRNA knockdown of p21 than after siRNA knockdown of p27. Our results imply that Cul4A may have a role in promoting cell renewal by avoiding G1 phase through regulation of both p21 and p27.
In summary, we have shown for the first time that the Cul4A gene is amplified and overexpressed in mesothelioma cells. Through shRNA knockdown and overexpression of ectopic Cul4A studies, we also showed that Cul4A controls the cell cycle and cell growth through p21 and p27 tumour suppressors in a p53-independent manner in mesothelioma cells. Our study demonstrates that the amplification of Cul4A may be an oncogenic event in mesothelioma development. Future studies on Cul4A as prognostic and therapeutic targets in MPM are warranted.