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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

Human phospho-kinase-profiler-array (HPKPA) revealed induction of phospho-p53(Ser15) in FBXW7- human CRC cells [FBXW7(−/−) vs. FBXW7(+/+)](A) Schematic representation of the coordinates of the pre-coated phospho-specific antibodies in duplicate in a nitrocellulose-membrane. Dashed red-circle highlights the antibody position for phospho-p53(Ser15). (B-E) Outcome overview of the HPKPA using human CRC-cells expressing and lacking FBXW7; FBXW7-deficient HCT116 (D) and DLD-1 (E) cells [FBXW7(−/−)], compared to parental HCT116 (B) and DLD-1 (C) cells [FBXW7(+/+)]. (F) The expression of phospho-p53(Ser15) protein was significantly increased. Scanned values obtained using transillumination scanner of phospho-p53(Ser15) and Positive Controls (PCs) spots as corresponding to HCT116 and DLD-1 cell lines in B-E. The results shown are representative of experiments performed at three independent times. (G) Phospho-p53(Ser15) expressions was analyzed by Western blotting analysis. Protein extracts isolated from HCT116 and DLD-1 cell lines with FBXW7(−/−) and FBXW7(+/+) alleles were Western blotted for phospho-p53(Ser15) antibody and Tubulin (loading control).
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Figure 1: Human phospho-kinase-profiler-array (HPKPA) revealed induction of phospho-p53(Ser15) in FBXW7- human CRC cells [FBXW7(−/−) vs. FBXW7(+/+)](A) Schematic representation of the coordinates of the pre-coated phospho-specific antibodies in duplicate in a nitrocellulose-membrane. Dashed red-circle highlights the antibody position for phospho-p53(Ser15). (B-E) Outcome overview of the HPKPA using human CRC-cells expressing and lacking FBXW7; FBXW7-deficient HCT116 (D) and DLD-1 (E) cells [FBXW7(−/−)], compared to parental HCT116 (B) and DLD-1 (C) cells [FBXW7(+/+)]. (F) The expression of phospho-p53(Ser15) protein was significantly increased. Scanned values obtained using transillumination scanner of phospho-p53(Ser15) and Positive Controls (PCs) spots as corresponding to HCT116 and DLD-1 cell lines in B-E. The results shown are representative of experiments performed at three independent times. (G) Phospho-p53(Ser15) expressions was analyzed by Western blotting analysis. Protein extracts isolated from HCT116 and DLD-1 cell lines with FBXW7(−/−) and FBXW7(+/+) alleles were Western blotted for phospho-p53(Ser15) antibody and Tubulin (loading control).

Mentions: Ablation of FBXW7 was shown to elevate the level of phosphorylated-substrate protein and its downstream signaling proteins. Such a phenomenon could inform about the disease mechanisms of colorectal carcinogenesis and the cellular pathways affected by homeostatic deregulation caused by an FBXW7 mutation. Post-translational modification of p53 by phosphorylation may be an important mechanism underlying regulation of p53 stabilization and function. However, the molecular and cellular mechanisms that link FBXW7 and p53 following phosphorylation are unclear. An in vitro human phospho-kinase array (HPKPA) with multiple p53-phosphoacceptor sites (Figure 1A), was used to assess changes to the protein phosphorylation profile. We and others have reported that HCT116 and DLD-1 cell-lines harboring wild-type FBXW7; FBXW7(+/+), or inactivated FBXW7; FBXW7(−/−), have the characteristics of human CRC cells without or with FBXW7-mutation [19, 20, 23, 28]. In particular, they provide an excellent in vitro model to delineate the molecular mechanisms that contribute to neoplasia. Remarkably, in the absence of FBXW7, both HCT116 and DLD-1 showed a substantial increase in p53 phosphorylation at Serine-15 as compared to control cells (Figure 1F), while phosphorylation at Serine-46 and Serine-392 remain unchanged (Figure 1, 1B vs. 1D and 1C vs. 1E). Western blot analysis showed an increase of p53 phosphorylated at Ser-15 in FBXW7(−/−) versus wild-type FBXW7(+/+) (Figure 1G).


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)

Human phospho-kinase-profiler-array (HPKPA) revealed induction of phospho-p53(Ser15) in FBXW7- human CRC cells [FBXW7(−/−) vs. FBXW7(+/+)](A) Schematic representation of the coordinates of the pre-coated phospho-specific antibodies in duplicate in a nitrocellulose-membrane. Dashed red-circle highlights the antibody position for phospho-p53(Ser15). (B-E) Outcome overview of the HPKPA using human CRC-cells expressing and lacking FBXW7; FBXW7-deficient HCT116 (D) and DLD-1 (E) cells [FBXW7(−/−)], compared to parental HCT116 (B) and DLD-1 (C) cells [FBXW7(+/+)]. (F) The expression of phospho-p53(Ser15) protein was significantly increased. Scanned values obtained using transillumination scanner of phospho-p53(Ser15) and Positive Controls (PCs) spots as corresponding to HCT116 and DLD-1 cell lines in B-E. The results shown are representative of experiments performed at three independent times. (G) Phospho-p53(Ser15) expressions was analyzed by Western blotting analysis. Protein extracts isolated from HCT116 and DLD-1 cell lines with FBXW7(−/−) and FBXW7(+/+) alleles were Western blotted for phospho-p53(Ser15) antibody and Tubulin (loading control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496214&req=5

Figure 1: Human phospho-kinase-profiler-array (HPKPA) revealed induction of phospho-p53(Ser15) in FBXW7- human CRC cells [FBXW7(−/−) vs. FBXW7(+/+)](A) Schematic representation of the coordinates of the pre-coated phospho-specific antibodies in duplicate in a nitrocellulose-membrane. Dashed red-circle highlights the antibody position for phospho-p53(Ser15). (B-E) Outcome overview of the HPKPA using human CRC-cells expressing and lacking FBXW7; FBXW7-deficient HCT116 (D) and DLD-1 (E) cells [FBXW7(−/−)], compared to parental HCT116 (B) and DLD-1 (C) cells [FBXW7(+/+)]. (F) The expression of phospho-p53(Ser15) protein was significantly increased. Scanned values obtained using transillumination scanner of phospho-p53(Ser15) and Positive Controls (PCs) spots as corresponding to HCT116 and DLD-1 cell lines in B-E. The results shown are representative of experiments performed at three independent times. (G) Phospho-p53(Ser15) expressions was analyzed by Western blotting analysis. Protein extracts isolated from HCT116 and DLD-1 cell lines with FBXW7(−/−) and FBXW7(+/+) alleles were Western blotted for phospho-p53(Ser15) antibody and Tubulin (loading control).
Mentions: Ablation of FBXW7 was shown to elevate the level of phosphorylated-substrate protein and its downstream signaling proteins. Such a phenomenon could inform about the disease mechanisms of colorectal carcinogenesis and the cellular pathways affected by homeostatic deregulation caused by an FBXW7 mutation. Post-translational modification of p53 by phosphorylation may be an important mechanism underlying regulation of p53 stabilization and function. However, the molecular and cellular mechanisms that link FBXW7 and p53 following phosphorylation are unclear. An in vitro human phospho-kinase array (HPKPA) with multiple p53-phosphoacceptor sites (Figure 1A), was used to assess changes to the protein phosphorylation profile. We and others have reported that HCT116 and DLD-1 cell-lines harboring wild-type FBXW7; FBXW7(+/+), or inactivated FBXW7; FBXW7(−/−), have the characteristics of human CRC cells without or with FBXW7-mutation [19, 20, 23, 28]. In particular, they provide an excellent in vitro model to delineate the molecular mechanisms that contribute to neoplasia. Remarkably, in the absence of FBXW7, both HCT116 and DLD-1 showed a substantial increase in p53 phosphorylation at Serine-15 as compared to control cells (Figure 1F), while phosphorylation at Serine-46 and Serine-392 remain unchanged (Figure 1, 1B vs. 1D and 1C vs. 1E). Western blot analysis showed an increase of p53 phosphorylated at Ser-15 in FBXW7(−/−) versus wild-type FBXW7(+/+) (Figure 1G).

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