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. 2014 Jun 1;15(6):768-76.
doi: 10.4161/cbt.28550. Epub 2014 Mar 18.

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

Joanna H M Tong  1 , Raymond W M Lung  1 , Frankie M C Sin  1 , Peggy P Y Law  1 , Wei Kang  1 , Anthony W H Chan  2 , Brigette B Y Ma  3 , Tony W C Mak  4 , Simon S M Ng  5 , Ka Fai To  1
Affiliations

Characterization of rare transforming KRAS mutations in sporadic colorectal cancer

Joanna H M Tong et al. Cancer Biol Ther. .

Abstract

KRAS mutational status has been shown to be a predictive biomarker of resistance to anti-EGFR monoclonal antibody (mAb) therapy in patients with metastatic colorectal cancer. We report the spectrum of KRAS mutation in 1506 patients with colorectal cancer and the identification and characterization of rare insertion mutations within the functional domain of KRAS. KRAS mutations are found in 44.5% (670/1506) of the patients. Two cases are found to harbor double mutations involving both codons 12 and 13. The frequencies of KRAS mutations at its codons 12, 13, 61, and 146 are 75.1%, 19.3%, 2.5%, and 2.7%, respectively. The most abundant mutation of codon 12 is G12D, followed by G12V and G12C while G13D is the predominant mutation in codon 13. Mutations in other codons are rare. The KRAS mutation rate is significantly higher in women (48%, 296/617) than in men (42.1%, 374/889, P = 0.023). Tumors on the right colon have a higher frequency of KRAS mutations than those on the left (57.3% vs. 40.4%, P<0.0001). Two in-frame insertion mutations affect the phosphate-binding loop (codon 10-16) of KRAS are identified. One of them has never been reported before. Compared with wild-type protein, the insertion variants enhance the cellular accumulation of active RAS (RAS-GTP) and constitutively activate the downstream signaling pathway. NIH3T3 cells transfected with the insertion variants show enhanced anchorage-independent growth and in vivo tumorigenicity. Potentially these mutations contribute to primary resistance to anti-EGFR mAb therapy but the clinical implication requires further validation.

Keywords: KRAS; colorectal cancer; targeted therapy.

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Figures

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Figure 1. Electropherogram for KRAS mutants. Tissue DNA from the patient with colorectal cancer were amplified and cloned for sequencing analysis. Two novel in-flame insertions (10G11 and 11GA12) in exon 2 of KRAS gene were identified.
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Figure 2.KRAS insertion mutants activated RAS signaling by enhancing cellular accumulation of active RAS (RAS-GTP) and activating p-ERK. NIH3T3 and 293FT cells were transfected with KRAS mutants, and RAS-GTP protein in the cell extract were immunoprecipitated with agarose beads containing Ras binding domain of Raf-1. Protein levels in both whole cell extracts (pan-RAS and pERK) and precipitated samples (RAS-GTP) were analyzed by western blot analysis as indicated. Representative results from 3 independent experiments were shown.
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Figure 3.KRAS insertion mutants promoted anchorage-independent growth in soft agar. NIH3T3 cells stably transfected with pcDNA3.1 empty vector (EV), wild-type KRAS (WT), G12V KRAS mutant (G12V), 10G11 and 11GA12 mutants were cultured in soft agar for analysis. Representative microscopic pictures of colony from each transfectant were taken (Magnification, 400×). The number of colony in each transfectant was plot in the bar chart and the results shown were mean and standard deviation from three independent experiments. The P value of < 0.05 and < 0.001 were denoted as * and ** respectively.
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Figure 4.KRAS insertion mutants promoted in vivo growth of NIH3T3 cells. In vivo tumorgenic assay in nude mice showed that tumors formed in the sites implanted with NIH3T3 cells expressing KRAS mutants (G12V, 10G11, or 11GA12) were consistently larger than that implanted with wild-type KRAS (WT) and empty vector (EV) controls. By western blotting, the expression of KRAS protein in the NIH3T3 transfectants and tumors dissected from the xenografts (T1–T5) was detected.
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