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A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition.

Fang Y, Wang Y, Wang Y, Meng Y, Zhu J, Jin H, Li J, Zhang D, Yu Y, Wu XR, Huang C - Biochem. J. (2014)

Bottom Line: Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure.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.The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.

View Article: PubMed Central - PubMed

Affiliation: ‡Department of Pathology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, China.

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.

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Inhibitory effect of p27Kip1 on JNK/c-Jun phosphorylation, EGFR expression and anchorage-independent growth in T24 and T24T cells(A) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then extracted for determination of protein expression by Western blotting. (B) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then subjected to determination of anchorage-independent growth in soft agar assay. The asterisk (*) indicates a significant decrease in anchorage-independent growth of T24T cells infected with Ad-GFP–p27 compared with those infected with Ad-GFP (P<0.01). (C) T24 cells were stably transfected with shRNA p27 and the cell extracts were subjected to Western blotting for determination of p27 knockdown in regulating JNK, p-c-Jun and EGFR expression, as compared with those transfected with control vector pLKO. (D) T24 cells were stably transfected with shRNA p27 or pLKO and then subjected to anchorage-independent growth. The asterisk (*) indicates a significant increase in anchorage-independent growth of T24 cells transfected with shRNA p27 compared with T24 cells transfected with pLKO (P<0.01).
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Figure 6: Inhibitory effect of p27Kip1 on JNK/c-Jun phosphorylation, EGFR expression and anchorage-independent growth in T24 and T24T cells(A) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then extracted for determination of protein expression by Western blotting. (B) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then subjected to determination of anchorage-independent growth in soft agar assay. The asterisk (*) indicates a significant decrease in anchorage-independent growth of T24T cells infected with Ad-GFP–p27 compared with those infected with Ad-GFP (P<0.01). (C) T24 cells were stably transfected with shRNA p27 and the cell extracts were subjected to Western blotting for determination of p27 knockdown in regulating JNK, p-c-Jun and EGFR expression, as compared with those transfected with control vector pLKO. (D) T24 cells were stably transfected with shRNA p27 or pLKO and then subjected to anchorage-independent growth. The asterisk (*) indicates a significant increase in anchorage-independent growth of T24 cells transfected with shRNA p27 compared with T24 cells transfected with pLKO (P<0.01).

Mentions: Compared with T24 cells, there was a reduction in p27Kip1 expression and up-regulation of EGFR expression in T24T cells (Figures 1A and 1B). To understand the biological consequences of p27Kip1-regulated EGFR expression, we also compared c-Jun and JNK activation as well as the anchorage-independent growth between T24 and T24T cells. As shown in Figure 5(A), the phosphorylation of JNK and c-Jun are both up-regulated in T24T cells. Consistently, an increase in anchorage-independent growth in T24T cells was also observed as compared with T24 cells (Figures 5B and 5C). Inhibition of JNK activation by pretreatment of T24T cells with the JNK inhibitor SP600125 blocked both c-Jun phosphorylation at Ser63/Ser73 and anchorage-independent growth of T24T cells (Figures 5D–5F). To provide direct evidence that p27Kip1-regulated EGFR expression via the JNK/c-Jun axis plays a role in cancer cell growth, we infected T24T cells with an adenovirus-driven-GFP–p27Kip1 and adenovirus-driven GFP as a negative control. Enforced expression of GFP–p27Kip1 in T24T cells dramatically inhibited the activation of JNK and c-Jun, expression of EGFR and anchorage-independent growth (Figures 6A and 6B). Moreover, knockdown of p27Kip1 in T24 cells led to an increased EGFR expression and anchorage-independent growth with induction of JNK and c-Jun activation (Figures 6C and 6D). Our results demonstrate that p27Kip1 down-regulation plays a key role in up-regulating the JNK/c-Jun pathway, leading to increased EGFR expression and anchorage-independent growth and endowing these cancer cells with a new metastatic potential.


A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition.

Fang Y, Wang Y, Wang Y, Meng Y, Zhu J, Jin H, Li J, Zhang D, Yu Y, Wu XR, Huang C - Biochem. J. (2014)

Inhibitory effect of p27Kip1 on JNK/c-Jun phosphorylation, EGFR expression and anchorage-independent growth in T24 and T24T cells(A) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then extracted for determination of protein expression by Western blotting. (B) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then subjected to determination of anchorage-independent growth in soft agar assay. The asterisk (*) indicates a significant decrease in anchorage-independent growth of T24T cells infected with Ad-GFP–p27 compared with those infected with Ad-GFP (P<0.01). (C) T24 cells were stably transfected with shRNA p27 and the cell extracts were subjected to Western blotting for determination of p27 knockdown in regulating JNK, p-c-Jun and EGFR expression, as compared with those transfected with control vector pLKO. (D) T24 cells were stably transfected with shRNA p27 or pLKO and then subjected to anchorage-independent growth. The asterisk (*) indicates a significant increase in anchorage-independent growth of T24 cells transfected with shRNA p27 compared with T24 cells transfected with pLKO (P<0.01).
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Related In: Results  -  Collection

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Figure 6: Inhibitory effect of p27Kip1 on JNK/c-Jun phosphorylation, EGFR expression and anchorage-independent growth in T24 and T24T cells(A) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then extracted for determination of protein expression by Western blotting. (B) T24T cells were infected with Ad-GFP or Ad-GFP–p27, and then subjected to determination of anchorage-independent growth in soft agar assay. The asterisk (*) indicates a significant decrease in anchorage-independent growth of T24T cells infected with Ad-GFP–p27 compared with those infected with Ad-GFP (P<0.01). (C) T24 cells were stably transfected with shRNA p27 and the cell extracts were subjected to Western blotting for determination of p27 knockdown in regulating JNK, p-c-Jun and EGFR expression, as compared with those transfected with control vector pLKO. (D) T24 cells were stably transfected with shRNA p27 or pLKO and then subjected to anchorage-independent growth. The asterisk (*) indicates a significant increase in anchorage-independent growth of T24 cells transfected with shRNA p27 compared with T24 cells transfected with pLKO (P<0.01).
Mentions: Compared with T24 cells, there was a reduction in p27Kip1 expression and up-regulation of EGFR expression in T24T cells (Figures 1A and 1B). To understand the biological consequences of p27Kip1-regulated EGFR expression, we also compared c-Jun and JNK activation as well as the anchorage-independent growth between T24 and T24T cells. As shown in Figure 5(A), the phosphorylation of JNK and c-Jun are both up-regulated in T24T cells. Consistently, an increase in anchorage-independent growth in T24T cells was also observed as compared with T24 cells (Figures 5B and 5C). Inhibition of JNK activation by pretreatment of T24T cells with the JNK inhibitor SP600125 blocked both c-Jun phosphorylation at Ser63/Ser73 and anchorage-independent growth of T24T cells (Figures 5D–5F). To provide direct evidence that p27Kip1-regulated EGFR expression via the JNK/c-Jun axis plays a role in cancer cell growth, we infected T24T cells with an adenovirus-driven-GFP–p27Kip1 and adenovirus-driven GFP as a negative control. Enforced expression of GFP–p27Kip1 in T24T cells dramatically inhibited the activation of JNK and c-Jun, expression of EGFR and anchorage-independent growth (Figures 6A and 6B). Moreover, knockdown of p27Kip1 in T24 cells led to an increased EGFR expression and anchorage-independent growth with induction of JNK and c-Jun activation (Figures 6C and 6D). Our results demonstrate that p27Kip1 down-regulation plays a key role in up-regulating the JNK/c-Jun pathway, leading to increased EGFR expression and anchorage-independent growth and endowing these cancer cells with a new metastatic potential.

Bottom Line: Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure.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.The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.

View Article: PubMed Central - PubMed

Affiliation: ‡Department of Pathology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, China.

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.

Show MeSH
Related in: MedlinePlus