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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 interacts with USP7A) Endogenous CCDC6 and USP7 proteins obtained from parental 293T cells were coimmunoprecipitated. The immunocomplexes were analysed by western blotting using the indicated antibodies. B) CCDC6 wt or the CCDC6 (1-223) deleted mutant constructs, transfected in 293T cells, were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. C) Immunoblot analysis of USP7 and of CCDC6 in NSCLC-derived cell lines: ADC derived cell lines (NCI-H1975, NCI-H460 and A549) and Large Cell Carcinoma cell line (NCI-H1299). Antitubulin is shown as loading control. D) Immunoblot analysis of CCDC6 expression in HeLa cells and in H460 cells overexpressing (+) FLAG-HA-USP7. Anti-FLAG immunoblot shows the USP7 transfection efficiency. Antitubulin is shown as loading control. E) 293T cells were transfected with myc-CCDC6 WT and TM constructs. Whole cell lysates (WCL) were prepared and equal amounts of proteins were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. F) Immunohistochemical analyses of human lung tumor tissues. Formalin fixed paraffin embedded lung tissue sections were stained with antibody against CCDC6 (a,b) or USP7 (c,d). Representative examples are shown of: CCDC6 low expression pattern (a), CCDC6 high expression pattern (b), USP7 low expression pattern (c), USP7 high expression pattern (d). Magnification: a, b, c, d x 200. G) Drug sensitivity to cisplatinum and olaparib in HCT116wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/−CCDC6+, and USP7−/−CCDC6+ was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, [CellTiter 96 AQueous One Solution assay (Promega)], as 50% inhibitory concentration (IC50) values. H) Combination index according to 1:2 concentration ratios of cisplatin and olaparib in HCT116 wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/− CCDC6+, USP7−/− CCDC6+ cells are shown. CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and antagonism, respectively.
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Figure 6: CCDC6 interacts with USP7A) Endogenous CCDC6 and USP7 proteins obtained from parental 293T cells were coimmunoprecipitated. The immunocomplexes were analysed by western blotting using the indicated antibodies. B) CCDC6 wt or the CCDC6 (1-223) deleted mutant constructs, transfected in 293T cells, were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. C) Immunoblot analysis of USP7 and of CCDC6 in NSCLC-derived cell lines: ADC derived cell lines (NCI-H1975, NCI-H460 and A549) and Large Cell Carcinoma cell line (NCI-H1299). Antitubulin is shown as loading control. D) Immunoblot analysis of CCDC6 expression in HeLa cells and in H460 cells overexpressing (+) FLAG-HA-USP7. Anti-FLAG immunoblot shows the USP7 transfection efficiency. Antitubulin is shown as loading control. E) 293T cells were transfected with myc-CCDC6 WT and TM constructs. Whole cell lysates (WCL) were prepared and equal amounts of proteins were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. F) Immunohistochemical analyses of human lung tumor tissues. Formalin fixed paraffin embedded lung tissue sections were stained with antibody against CCDC6 (a,b) or USP7 (c,d). Representative examples are shown of: CCDC6 low expression pattern (a), CCDC6 high expression pattern (b), USP7 low expression pattern (c), USP7 high expression pattern (d). Magnification: a, b, c, d x 200. G) Drug sensitivity to cisplatinum and olaparib in HCT116wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/−CCDC6+, and USP7−/−CCDC6+ was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, [CellTiter 96 AQueous One Solution assay (Promega)], as 50% inhibitory concentration (IC50) values. H) Combination index according to 1:2 concentration ratios of cisplatin and olaparib in HCT116 wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/− CCDC6+, USP7−/− CCDC6+ cells are shown. CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and antagonism, respectively.

Mentions: USP7 is the de-ubiquitinase (DUB) predicted to remove ubiquitin from CCDC6 [21]. We verified that endogenous CCDC6 interacts specifically with USP7 (Figure 6A). The myc-tagged CCDC6 truncated mutant (1-223) still coimmunoprecipitated with USP7, suggesting that the aminoterminus of CCDC6 is sufficient for the CCDC6 binding to USP7 (Figure 6B). Recent findings unveiled that in NSCLC cell lines, H1975, H1299, A549 and H460, CCDC6 transcripts did not show significant variability, while quite different CCDC6 protein levels were observed [14]. Moreover, by cycloheximide experiments differences of the CCDC6 half life were detected in the NSCLC cells (Supplementary Figure 3).


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 interacts with USP7A) Endogenous CCDC6 and USP7 proteins obtained from parental 293T cells were coimmunoprecipitated. The immunocomplexes were analysed by western blotting using the indicated antibodies. B) CCDC6 wt or the CCDC6 (1-223) deleted mutant constructs, transfected in 293T cells, were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. C) Immunoblot analysis of USP7 and of CCDC6 in NSCLC-derived cell lines: ADC derived cell lines (NCI-H1975, NCI-H460 and A549) and Large Cell Carcinoma cell line (NCI-H1299). Antitubulin is shown as loading control. D) Immunoblot analysis of CCDC6 expression in HeLa cells and in H460 cells overexpressing (+) FLAG-HA-USP7. Anti-FLAG immunoblot shows the USP7 transfection efficiency. Antitubulin is shown as loading control. E) 293T cells were transfected with myc-CCDC6 WT and TM constructs. Whole cell lysates (WCL) were prepared and equal amounts of proteins were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. F) Immunohistochemical analyses of human lung tumor tissues. Formalin fixed paraffin embedded lung tissue sections were stained with antibody against CCDC6 (a,b) or USP7 (c,d). Representative examples are shown of: CCDC6 low expression pattern (a), CCDC6 high expression pattern (b), USP7 low expression pattern (c), USP7 high expression pattern (d). Magnification: a, b, c, d x 200. G) Drug sensitivity to cisplatinum and olaparib in HCT116wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/−CCDC6+, and USP7−/−CCDC6+ was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, [CellTiter 96 AQueous One Solution assay (Promega)], as 50% inhibitory concentration (IC50) values. H) Combination index according to 1:2 concentration ratios of cisplatin and olaparib in HCT116 wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/− CCDC6+, USP7−/− CCDC6+ cells are shown. CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and antagonism, respectively.
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Figure 6: CCDC6 interacts with USP7A) Endogenous CCDC6 and USP7 proteins obtained from parental 293T cells were coimmunoprecipitated. The immunocomplexes were analysed by western blotting using the indicated antibodies. B) CCDC6 wt or the CCDC6 (1-223) deleted mutant constructs, transfected in 293T cells, were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. C) Immunoblot analysis of USP7 and of CCDC6 in NSCLC-derived cell lines: ADC derived cell lines (NCI-H1975, NCI-H460 and A549) and Large Cell Carcinoma cell line (NCI-H1299). Antitubulin is shown as loading control. D) Immunoblot analysis of CCDC6 expression in HeLa cells and in H460 cells overexpressing (+) FLAG-HA-USP7. Anti-FLAG immunoblot shows the USP7 transfection efficiency. Antitubulin is shown as loading control. E) 293T cells were transfected with myc-CCDC6 WT and TM constructs. Whole cell lysates (WCL) were prepared and equal amounts of proteins were immunoprecipitated with anti-myc. Then, the immunocomplexes were analysed by western blot using the indicated antibodies. F) Immunohistochemical analyses of human lung tumor tissues. Formalin fixed paraffin embedded lung tissue sections were stained with antibody against CCDC6 (a,b) or USP7 (c,d). Representative examples are shown of: CCDC6 low expression pattern (a), CCDC6 high expression pattern (b), USP7 low expression pattern (c), USP7 high expression pattern (d). Magnification: a, b, c, d x 200. G) Drug sensitivity to cisplatinum and olaparib in HCT116wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/−CCDC6+, and USP7−/−CCDC6+ was determined by a modified 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide assay, [CellTiter 96 AQueous One Solution assay (Promega)], as 50% inhibitory concentration (IC50) values. H) Combination index according to 1:2 concentration ratios of cisplatin and olaparib in HCT116 wt, FBXW7−/−, CCDC6−/−, USP7−/−, CCDC6−/− CCDC6+, USP7−/− CCDC6+ cells are shown. CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and antagonism, respectively.
Mentions: USP7 is the de-ubiquitinase (DUB) predicted to remove ubiquitin from CCDC6 [21]. We verified that endogenous CCDC6 interacts specifically with USP7 (Figure 6A). The myc-tagged CCDC6 truncated mutant (1-223) still coimmunoprecipitated with USP7, suggesting that the aminoterminus of CCDC6 is sufficient for the CCDC6 binding to USP7 (Figure 6B). Recent findings unveiled that in NSCLC cell lines, H1975, H1299, A549 and H460, CCDC6 transcripts did not show significant variability, while quite different CCDC6 protein levels were observed [14]. Moreover, by cycloheximide experiments differences of the CCDC6 half life were detected in the NSCLC cells (Supplementary Figure 3).

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