Abstract
p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
Highlights
P27Kip1, encoded by the CDKN1B gene, is a strong inhibitor of the cyclin-dependent kinase (CDK) that propel the progression of the cell cycle from G1- to S-phase [1]
Of the differentially expressed proteins, we found epidermal growth factor receptor (EGFR) and p27Kip1 to be among the most prominent. p27Kip1 expression was dramatically down-regulated in T24T cells compared with its parental T24 cells, whereas EGFR was exactly the opposite, i.e. low expression in T24 cells and high in T24T cells (Figure 1A)
To further extend our findings to specimens obtained from human cancer patients, we evaluated the expression of both p27Kip1 and EGFR in human cancer specimens using IHC staining
Summary
P27Kip, encoded by the CDKN1B gene, is a strong inhibitor of the CDKs (cyclin-dependent kinases) that propel the progression of the cell cycle from G1- to S-phase [1]. The results of the present study expand the existing repertoire of CDKindependent activities of p27Kip, and suggest that the increased cell-cycle transition due to p27Kip deficiency can synergize with increased expression of growth-promoting signals (e.g. EGFR) to accelerate tumour progression. Immortalized p27Kip1 + / + and p27Kip1 − / − MEF (mouse embryonic fibroblast) cell lines have been described in our previous studies [19,22,23]. These cells and their stable transfectants were maintained at 37 ◦C in a 5 % CO2 incubator with DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10 % FBS, 2 μM L-glutamine and 25 μg/ml gentamycin.
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