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FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15.

Li N, Lorenzi F, Kalakouti E, Normatova M, Babaei-Jadidi R, Tomlinson I, Nateri AS - Oncotarget (2015)

Bottom Line: Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes.Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells.Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genetics & Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham NG7 2UH, UK.

ABSTRACT
FBXW7 mutations occur in a variety of human cancers including colorectal cancer (CRC). Elucidating its mechanism of action has become crucial for cancer therapy; however, it is also complicated by the fact that FBXW7 can influence many pathways due to its role as an E3-ubiquitin ligase in proteasome degradation. FBXW7 and TP53 are tumour suppressors intensively implicated in colorectal carcinogenesis. Deletion mutations in these two genes in animal models mark the progression from adenoma to carcinoma. Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes. The underlying mechanism requires further investigation. In our laboratory, we have used a phospho-kinase profiler array to illustrate a potential molecular link between FBXW7 and p53 in CRC cells. In vitro and in vivo assessments demonstrated aberrant induction of phosphorylated p53 at Serine 15 [phospho-p53(Ser15)] in human FBXW7-deficient CRC cells as compared to their FBXW7-wild-type counterparts. FBXW7 loss in HCT116 cells promoted resistance to oxaliplatin. Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells. The findings may suggest the applicability of phospho-p53(Ser15) as an indicative marker of FBXW7-mutations. Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.

No MeSH data available.


Related in: MedlinePlus

phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction(A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116FBXW7(+/+) versus HCT116FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116FBXW7(−/−) compared with parental HCT116FBXW7(+/+) cells treated with the oxaliplatin.
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Figure 8: phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction(A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116FBXW7(+/+) versus HCT116FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116FBXW7(−/−) compared with parental HCT116FBXW7(+/+) cells treated with the oxaliplatin.

Mentions: In the present studies, we demonstrated through several lines of evidence that FBXW7 ablation in CRC cells results in the accumulation of phospho-p53(Ser15). The mechanism of this is still uncertain. We therefore studied the FBXW7/p53-correlation further to validate the phospho-p53(Ser15) phenomenon. Consistent with the HPKPA results, mutations at either Ser46 (S46A) or Ser392 (S392A) did not stabilize phospho-p53(Ser15) in the presence of FBXW7 (Figure 8A). Western blotting data with cells that overexpress the p53(S15A) mutant protein also determined the anti-phospho-p53(Ser15) antibody specificity (Figure 8A). Furthermore, co-expression of FBXW7 decreased the steady-state levels of p53(wt) (Figure 8B). We next examined whether the FBXW7 protein would bind to p53 protein and vice versa. Immunoprecipitation (PI) of either FLAG epitope-tagged p53 or GFP fused-FBXW7α showed no interaction between these proteins (Figure 8C). These data suggest that FBXW7 does not act directly, but indirectly regulates the abundance of phospho-p53(Ser15).


FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15.

Li N, Lorenzi F, Kalakouti E, Normatova M, Babaei-Jadidi R, Tomlinson I, Nateri AS - Oncotarget (2015)

phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction(A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116FBXW7(+/+) versus HCT116FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116FBXW7(−/−) compared with parental HCT116FBXW7(+/+) cells treated with the oxaliplatin.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction(A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116FBXW7(+/+) versus HCT116FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116FBXW7(−/−) compared with parental HCT116FBXW7(+/+) cells treated with the oxaliplatin.
Mentions: In the present studies, we demonstrated through several lines of evidence that FBXW7 ablation in CRC cells results in the accumulation of phospho-p53(Ser15). The mechanism of this is still uncertain. We therefore studied the FBXW7/p53-correlation further to validate the phospho-p53(Ser15) phenomenon. Consistent with the HPKPA results, mutations at either Ser46 (S46A) or Ser392 (S392A) did not stabilize phospho-p53(Ser15) in the presence of FBXW7 (Figure 8A). Western blotting data with cells that overexpress the p53(S15A) mutant protein also determined the anti-phospho-p53(Ser15) antibody specificity (Figure 8A). Furthermore, co-expression of FBXW7 decreased the steady-state levels of p53(wt) (Figure 8B). We next examined whether the FBXW7 protein would bind to p53 protein and vice versa. Immunoprecipitation (PI) of either FLAG epitope-tagged p53 or GFP fused-FBXW7α showed no interaction between these proteins (Figure 8C). These data suggest that FBXW7 does not act directly, but indirectly regulates the abundance of phospho-p53(Ser15).

Bottom Line: Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes.Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells.Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genetics & Stem Cell Group, Cancer Biology Unit, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham NG7 2UH, UK.

ABSTRACT
FBXW7 mutations occur in a variety of human cancers including colorectal cancer (CRC). Elucidating its mechanism of action has become crucial for cancer therapy; however, it is also complicated by the fact that FBXW7 can influence many pathways due to its role as an E3-ubiquitin ligase in proteasome degradation. FBXW7 and TP53 are tumour suppressors intensively implicated in colorectal carcinogenesis. Deletion mutations in these two genes in animal models mark the progression from adenoma to carcinoma. Although still largely unknown, the last defense mechanism against CRC at the molecular level could be through a synergistic effect of the two genes. The underlying mechanism requires further investigation. In our laboratory, we have used a phospho-kinase profiler array to illustrate a potential molecular link between FBXW7 and p53 in CRC cells. In vitro and in vivo assessments demonstrated aberrant induction of phosphorylated p53 at Serine 15 [phospho-p53(Ser15)] in human FBXW7-deficient CRC cells as compared to their FBXW7-wild-type counterparts. FBXW7 loss in HCT116 cells promoted resistance to oxaliplatin. Immunoblotting data further confirmed that reduction of phospho-p53(Ser15) may contribute to the decreased efficacy of therapy in FBXW7-mutated CRC cells. The findings may suggest the applicability of phospho-p53(Ser15) as an indicative marker of FBXW7-mutations. Phospho-p53(Ser15) regulation by FBXW7 E3-ligase activity could provide important clues for understanding FBXW7 behavior in tumour progression and grounds for its clinical applicability thereafter.

No MeSH data available.


Related in: MedlinePlus