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FBXW7 and USP7 regulate CCDC6 turnover during the cell cycle and affect cancer drugs susceptibility in NSCLC.

Morra F, Luise C, Merolla F, Poser I, Visconti R, Ilardi G, Paladino S, Inuzuka H, Guggino G, Monaco R, Colecchia D, Monaco G, Cerrato A, Chiariello M, Denning K, Claudio PP, Staibano S, Celetti A - Oncotarget (2015)

Bottom Line: CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis.The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl.The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

View Article: PubMed Central - PubMed

Affiliation: Istituto per l'Endocrinologia e l'Oncologia Sperimentale "Gaetano Salvatore", CNR, Napoli, Italy.

ABSTRACT
CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

No MeSH data available.


Related in: MedlinePlus

CCDC6 protein levels are regulated through the cell cycleA) CCDC6 levels increase from G1 to mitosis. Human HeLa cells were synchronized at the G1/S boundary by double thymidine block (TT block), then released into the cell cycle. Samples were analysed by SDS-PAGE and immunoblotted using the specific antibodies at the times shown after release from the block. B) HeLa cells were synchronized at the G1/S boundary by TT block, at the G2 phase by the CDK1 inhibitor RO3306, in mitosis after treatment with 100 ng/ml nocodazole (Noco) for 16 hours. Floating cells were separated from adherent and samples were analysed by immunoblot using the anti-CCDC6 antibody. C) Human HeLa cells were treated with 100 ng/ml nocodazole for 16 hours. Floating, mitotic cells, were separated from adherent (interphase) cells, plated again and samples were taken at the indicated times and analysed by immunoblotting using the indicated antibodies. D) HeLa cells were synchronized as in C and collected at time 0. Extracts were treated in vitro with CIP, as indicated. Therefore, samples were taken and analysed by immunoblotting with the indicated antibodies. Anti-MPM2 is utilized as indicator of mitotic arrest. E) HeLa cells were synchronized as in C, and cells were treated with RO3306 (9 μM for 2 hours) or with SB216763 (10 μM for 4 hours) before the nocodazole release, as indicated. Samples were analysed by SDS-PAGE and immunoblotted using the indicated antibodies.
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Figure 1: CCDC6 protein levels are regulated through the cell cycleA) CCDC6 levels increase from G1 to mitosis. Human HeLa cells were synchronized at the G1/S boundary by double thymidine block (TT block), then released into the cell cycle. Samples were analysed by SDS-PAGE and immunoblotted using the specific antibodies at the times shown after release from the block. B) HeLa cells were synchronized at the G1/S boundary by TT block, at the G2 phase by the CDK1 inhibitor RO3306, in mitosis after treatment with 100 ng/ml nocodazole (Noco) for 16 hours. Floating cells were separated from adherent and samples were analysed by immunoblot using the anti-CCDC6 antibody. C) Human HeLa cells were treated with 100 ng/ml nocodazole for 16 hours. Floating, mitotic cells, were separated from adherent (interphase) cells, plated again and samples were taken at the indicated times and analysed by immunoblotting using the indicated antibodies. D) HeLa cells were synchronized as in C and collected at time 0. Extracts were treated in vitro with CIP, as indicated. Therefore, samples were taken and analysed by immunoblotting with the indicated antibodies. Anti-MPM2 is utilized as indicator of mitotic arrest. E) HeLa cells were synchronized as in C, and cells were treated with RO3306 (9 μM for 2 hours) or with SB216763 (10 μM for 4 hours) before the nocodazole release, as indicated. Samples were analysed by SDS-PAGE and immunoblotted using the indicated antibodies.

Mentions: To investigate the regulation of CCDC6 protein levels during the cell cycle, human HeLa cells were synchronised at the G1/S boundary using a double thymidine (TT) block, then released into the cell cycle. The time course experiment showed several post-translational modifications of CCDC6 protein at different time points (Figure 1A). In order to better discern the CCDC6 behaviour during the cell cycle, we utilized different chemical treatments to block the cells in specific phases. We observed a cyclic variation of CCDC6 levels that increased at boundary G1/S, picked at G2, and appeared upshifted in mitotic phase (Figure 1B). In order to focus on the CCDC6 modifications in mitosis, HeLa cells were then treated with nocodazole for 16 h, collected by shake-off, plated again and analyzed at different time points for a mitotic time course. The product of CCDC6 appeared as multiple bands on SDS-page gels at time 0, from the nocodazole release. The shifts were maintained up to 2 hours, while they disappeared at 4, 8 and 10 hours when the protein seemed to be stabilized (Figure 1C). Calf intestinal phosphatase (CIP) treatment determined a significant decrease of the shifted bands at time 0, thus suggesting that the modifications observed in mitosis might depend on phosphorylation events (Figure 1D). The specific inhibition of cyclin–dependent kinase 1 (CDK1) by RO3306 reverted most of the shifts observed by electrophoresis. In addition, the pretreatment with the specific inhibitor of glycogen synthase kinase 3 (GSK3), SB216763, also reverted the modifications detected for CCDC6 in mitosis (Figure 1E).


FBXW7 and USP7 regulate CCDC6 turnover during the cell cycle and affect cancer drugs susceptibility in NSCLC.

Morra F, Luise C, Merolla F, Poser I, Visconti R, Ilardi G, Paladino S, Inuzuka H, Guggino G, Monaco R, Colecchia D, Monaco G, Cerrato A, Chiariello M, Denning K, Claudio PP, Staibano S, Celetti A - Oncotarget (2015)

CCDC6 protein levels are regulated through the cell cycleA) CCDC6 levels increase from G1 to mitosis. Human HeLa cells were synchronized at the G1/S boundary by double thymidine block (TT block), then released into the cell cycle. Samples were analysed by SDS-PAGE and immunoblotted using the specific antibodies at the times shown after release from the block. B) HeLa cells were synchronized at the G1/S boundary by TT block, at the G2 phase by the CDK1 inhibitor RO3306, in mitosis after treatment with 100 ng/ml nocodazole (Noco) for 16 hours. Floating cells were separated from adherent and samples were analysed by immunoblot using the anti-CCDC6 antibody. C) Human HeLa cells were treated with 100 ng/ml nocodazole for 16 hours. Floating, mitotic cells, were separated from adherent (interphase) cells, plated again and samples were taken at the indicated times and analysed by immunoblotting using the indicated antibodies. D) HeLa cells were synchronized as in C and collected at time 0. Extracts were treated in vitro with CIP, as indicated. Therefore, samples were taken and analysed by immunoblotting with the indicated antibodies. Anti-MPM2 is utilized as indicator of mitotic arrest. E) HeLa cells were synchronized as in C, and cells were treated with RO3306 (9 μM for 2 hours) or with SB216763 (10 μM for 4 hours) before the nocodazole release, as indicated. Samples were analysed by SDS-PAGE and immunoblotted using the indicated antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494967&req=5

Figure 1: CCDC6 protein levels are regulated through the cell cycleA) CCDC6 levels increase from G1 to mitosis. Human HeLa cells were synchronized at the G1/S boundary by double thymidine block (TT block), then released into the cell cycle. Samples were analysed by SDS-PAGE and immunoblotted using the specific antibodies at the times shown after release from the block. B) HeLa cells were synchronized at the G1/S boundary by TT block, at the G2 phase by the CDK1 inhibitor RO3306, in mitosis after treatment with 100 ng/ml nocodazole (Noco) for 16 hours. Floating cells were separated from adherent and samples were analysed by immunoblot using the anti-CCDC6 antibody. C) Human HeLa cells were treated with 100 ng/ml nocodazole for 16 hours. Floating, mitotic cells, were separated from adherent (interphase) cells, plated again and samples were taken at the indicated times and analysed by immunoblotting using the indicated antibodies. D) HeLa cells were synchronized as in C and collected at time 0. Extracts were treated in vitro with CIP, as indicated. Therefore, samples were taken and analysed by immunoblotting with the indicated antibodies. Anti-MPM2 is utilized as indicator of mitotic arrest. E) HeLa cells were synchronized as in C, and cells were treated with RO3306 (9 μM for 2 hours) or with SB216763 (10 μM for 4 hours) before the nocodazole release, as indicated. Samples were analysed by SDS-PAGE and immunoblotted using the indicated antibodies.
Mentions: To investigate the regulation of CCDC6 protein levels during the cell cycle, human HeLa cells were synchronised at the G1/S boundary using a double thymidine (TT) block, then released into the cell cycle. The time course experiment showed several post-translational modifications of CCDC6 protein at different time points (Figure 1A). In order to better discern the CCDC6 behaviour during the cell cycle, we utilized different chemical treatments to block the cells in specific phases. We observed a cyclic variation of CCDC6 levels that increased at boundary G1/S, picked at G2, and appeared upshifted in mitotic phase (Figure 1B). In order to focus on the CCDC6 modifications in mitosis, HeLa cells were then treated with nocodazole for 16 h, collected by shake-off, plated again and analyzed at different time points for a mitotic time course. The product of CCDC6 appeared as multiple bands on SDS-page gels at time 0, from the nocodazole release. The shifts were maintained up to 2 hours, while they disappeared at 4, 8 and 10 hours when the protein seemed to be stabilized (Figure 1C). Calf intestinal phosphatase (CIP) treatment determined a significant decrease of the shifted bands at time 0, thus suggesting that the modifications observed in mitosis might depend on phosphorylation events (Figure 1D). The specific inhibition of cyclin–dependent kinase 1 (CDK1) by RO3306 reverted most of the shifts observed by electrophoresis. In addition, the pretreatment with the specific inhibitor of glycogen synthase kinase 3 (GSK3), SB216763, also reverted the modifications detected for CCDC6 in mitosis (Figure 1E).

Bottom Line: CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis.The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl.The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

View Article: PubMed Central - PubMed

Affiliation: Istituto per l'Endocrinologia e l'Oncologia Sperimentale "Gaetano Salvatore", CNR, Napoli, Italy.

ABSTRACT
CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

No MeSH data available.


Related in: MedlinePlus