Serine substitution of proline at codon 151 of TP53 confers gain of function activity leading to anoikis resistance and tumor progression of head and neck cancer cells - PubMed (original) (raw)

Comparative Study

. 2013 Jun;123(6):1416-23.

doi: 10.1002/lary.23846. Epub 2013 Apr 26.

Affiliations

Comparative Study

Serine substitution of proline at codon 151 of TP53 confers gain of function activity leading to anoikis resistance and tumor progression of head and neck cancer cells

Tong-Xin Xie et al. Laryngoscope. 2013 Jun.

Abstract

Objectives/hypothesis: Mutation of the TP53 gene occurs in more than half of cases of head and neck squamous cell carcinoma (HNSCC). However, little is known about how specific TP53 mutations affect tumor progression. The objective of this study is to determine the gain of function of mutant p53 with a proline-to-serine substitution at codon 151.

Study design: Laboratory-based study.

Methods: A panel of HNSCC cell lines was determined with anoikis assays, and orthotopic mouse experiments were performed. TP53 was sequenced. The shRNA knockdown and overexpression approaches were used for testing mutant p53 functions. The crystal structure of the p53 protein was analyzed using an in silico approach.

Results: An anoikis-resistant cell line, Tu138, was found to have a proline-to-serine substitution at codon 151 of TP53, which results in loss of wild-type p53 transcriptional activity. Moreover, the mutant p53 was shown to promote anoikis resistance and soft agar growth. Using an in silico approach based on the crystal structure of wild-type p53 protein, substitution of proline by serine at position 151 would create a cavity in a hydrophobic pocket, the loss of van der Waals contacts, and the thermodynamically unfavorable placement of a polar group, the hydroxyl oxygen atom of the serine, within a hydrophobic region, all of which likely cause a locally altered structure.

Conclusions: Our data suggest that mutation at position 151 leads to a structural alteration, which results in significant functional changes in the p53 protein that impact tumor progression.

Copyright © 2012 The American Laryngological, Rhinological and Otological Society, Inc.

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Conflict of interest statement

The authors have no other funding, financial relationships, or conflicts of interest to disclose.

Figures

Figure 1

Figure 1

The top 8 anoikis-resistant and 8 anoikis sensitive cell lines were selected and injected into the tongues of nude mice. The association of anoikis resistance (detached for 48 hrs) with tumor size at 32 days after injection was analyzed.

Figure 2

Figure 2

A. Tu138 and UMSCC1 cells were cultured in detached conditions for 24 and 48 hrs and dead cells were determined using Trypan-blue (** p<0.01, n=3). B. Tu138 and UMSCC1 cells were detached for 6, 12 and 24 hrs and fragmented DNA was isolated and subjected to agarose gel electrophoresis. C. The p53 expression in Tu138 and UMSCC1 cells was determined with Western blot after normal and detached culture for 24 hrs. D. Tu138, UMSCC1 and UMSCC1c2 cells were treated with 5ug/ml 5-FU for 20 hrs and the wild-type p53 MCF-7 cells were used as control. The p53 target genes p21 and MDM2 were examined using Western blotting.

Figure 3

Figure 3

p53P151S makes fewer inter residue contacts than wild type p53. (Left) Structure of wild type p53 in the vicinity of residue P151, Residue P151 and nearby interacting amino acid residues are shown as sticks and colored according to atom type whereby oxygen is red, nitrogen, blue and carbon, white. Dashed yellow lines indicate contacts between residue P151 and nearby residues (d ≤ 4.2 Å). (Right) The structure of the modeled p53P151S mutant, colored and demarked as in the left panel. The closest approach of the side chains of S151 and V147 is 5.8 Å.

Figure 4

Figure 4

P53P151S can confer anoikis resistance. Western blot was performed to detect p53 knockdown effect in stable Tu138 cells after normal and detached culture for 24 hrs. B. Tu138 p53 knockdown cells and vector control cells were detached for 24 and 48 hrs and dead cells were determined with Trypan Blue (**, p<0.01, n=3). C. UMSCC1 cells were infected with retroviral vector pBaBe containing p53P151S cloned from Tu138 cells and empty vector infected cells as control. The stable cells were cultured in attached and detached conditions for 24 hrs. D. Anoikis assay was performed in UMSCC1 cells expressing p53P151S and their control cells (**, p<0.01, n=3).

Figure 5

Figure 5

Mutant p53P151S promotes cell growth in soft agar. P53-knockdown Tu138 cells and UMSCC1 cells overexpressing mutant p53 and their control cells were cultured in soft agar for 14 days (for Tu138 cells, A and B. **, p<0.01, n=3) and 21 days (for UMSCC1cells, 5C and D. **, p<0.01, n=3).

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