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. 2010 Dec 31;5(12):e16012.
doi: 10.1371/journal.pone.0016012.

Nonsense mediated decay resistant mutations are a source of expressed mutant proteins in colon cancer cell lines with microsatellite instability (VSports注册入口)

David S Williams  1 , Matthew J Bird, Robert N Jorissen, Yen Lin Yu, Francesca Walker, Hui Hua Zhang, Edouard C Nice, Antony W Burgess
Affiliations

Nonsense mediated decay resistant mutations are a source of expressed mutant proteins in colon cancer cell lines with microsatellite instability

David S Williams et al. PLoS One. .

Erratum in

  • PLoS One. 2011;6(1). doi: 10.1371/annotation/53805ecf-7d10-4d99-9cec-f27f5e0d4166. Walker, Franscesa [corrected to Walker, Francesca]

Abstract

Background: Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD) VSports手机版. NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours. .

Methods: Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines V体育安卓版. .

Results: Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested. V体育ios版.

Conclusion: NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological targets in a vaccine targeting MSI-High colonic tumours. VSports最新版本.

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Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of normal BAX protein, but not mutant BAX protein by MSI-High cell lines.
(A) Normal BAX protein (21 kDa, arrow), but no mutant BAX protein (predicted 6.4 kDa) was detected in Western Blot analyses on whole cell lysates of colon cancer cell lines. Cell lines with BAX G8 cMS mutation status: 1. LoVo (−1, +1), 2. SW480 (wt), 3. HCA7 (−1, wt), 4. LS174T (−1), 5. HCT116 (−1, wt), 6. HeLa (wt), 7. LIM1215 (−1). (B) Homozygous −1 base mutation in G8 cMS in LS174T cell line (top) compared to non-mutated cell line, SW1222 (bottom) (reverse DNA sequencing). (C) Normal BAX protein (arrow), but no mutant BAX protein detected by immunoprecipitation: 1. HeLa (wt), 2. LS174T (−1). IgG light chain at 25 kDa.
Figure 2
Figure 2. Expression of mutant CREBBP and EP300 proteins, arising from NMD-resistant mutant transcripts.
Normal proteins (white arrow) and mutant proteins (black arrow) were detected in Western Blot analyses on whole cell lysates for (A) CREBBP and (B) EP300. (A) CREBBP C5 cMS: 1. SW480 (wt), 2. LoVo (−1, wt), 3. HCT116 (wt), 4. LIM1215 (wt), 5. LS174T (wt). MW (kDa): wt 265, mut 209. (B) EP300 A5 cMS: 1. LoVo (wt), 2. HCT116 (−1, wt), 3. HCA7 (wt), 4. LIM1215 (wt), 5. LS174T (wt). MW (kDa): wt 223, mut 187.
Figure 3
Figure 3. Validation of mutant CREBBP protein.
Specificity of the mutant protein band was confirmed by gene-specific knockdown in transfectants of siRNA targeting CREBBP. (A) Detection of mutant protein band (black arrow) in whole cell lysates of LoVo cell line, but normal protein only (white arrow) in non-mutated SW480 cell line. (B) Western Blot of LoVo whole cell lysates from siRNA SMARTpool transfectants show decreased expression of normal (white arrow) and mutant (black arrow) CREBBP protein band in CREBBP-targeted lysates (lane 1). Beta-catenin loading control (grey arrow) 92 kDa. (C) Relative intensity of normal (blue) and mutant (red) CREBBP protein bands compared to untransfected cells (PBS). (D) Decreased CREBBP mRNA expression in CREBBP siRNA transfectants (left lane) relative to untransfected cells (right lane).
Figure 4
Figure 4. Validation of mutant EP300 protein.
Specificity of the mutant protein band confirmed by gene-specific knockdown in transfectants of siRNA targeting EP300. (A) qRT-PCR data to assess siRNAs targeting EP300 (best knockdown using P300-1 and P300-3). (B–C) Decreased mutant EP300 protein detected in Western Blot analyses on immunoprecipitates of EP300 siRNA transfectants relative to untransfected controls (PBS).
Figure 5
Figure 5. Potential mutant TTK and AIM2 proteins.
(A) Western blot analyses on whole cell lysates identified a potential mutant TTK protein band (black arrow) in multiple mutated cell lines and normal TTK protein (white arrow) only in non-mutated lines: 1. LIM1863 (wt), 2. HCA7 (wt), 3. LIM1899 (−1, wt), 4. HCT116 (−1, wt), 5. LoVo (−1, wt), 6. LIM2537 (−1, wt), 7. LIM2551 (−1, wt). TTK MW (kDa): wt 97, mut 101. (B) Western blot analyses on immunoprecipitates of whole cell lysates identified an AIM2 protein band (black arrow) in all cell lines, including the homozygous mutant LoVo cell line: 1. SW480 (wt), 2. LoVo (−1), 3. HCT116 (−1, wt). 4, LS174T (wt). AIM2 MW (kDa): wt 39.0, mut 40.4.
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References

    1. Woerner SM, Benner A, Sutter C, Schiller M, Yuan YP, et al. Pathogenesis of DNA repair-deficient cancers: a statistical meta-analysis of putative Real Common Target genes. Oncogene. 2003;22:2226–2235. - PubMed
    1. Duval A, Hamelin R. Genetic instability in human mismatch repair deficient cancers. Ann Genet. 2002;45:71–75. - PubMed
    1. Niv Y. Microsatellite instability and MLH1 promoter hypermethylation in colorectal cancer. World J Gastroenterol. 2007;13:1767–1769. - PMC (V体育平台登录) - PubMed
    1. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248–5257. - "V体育官网" PubMed
    1. Jenkins MA, Hayashi S, O'Shea AM, Burgart LJ, Smyrk TC, et al. Pathology features in Bethesda guidelines predict colorectal cancer microsatellite instability: a population-based study. Gastroenterology. 2007;133:48–56. - VSports - PMC - PubMed

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