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Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells.

ABSTRACT: Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers.

SUBMITTER: Waters AM

PROVIDER: S-EPMC5042562 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells.

Waters Andrew M AM Bagni Rachel R Portugal Franklin F Hartley James L JL

PloS one 20160929 9

Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were ...[more]

PMID: 27684555

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Project description:In a number of types of cancer, anoikis, a form of apoptosis induced by loss of extracellular matrix (ECM) attachment, is disturbed. Anoikis resistance is essential in the formation of metastases. A recent study identified carcinoma cell subpopulations surviving without ECM contact in pathological specimens of colorectal cancer. The occurrence of these subpopulations indicated anoikis resistance. In the present study, it is demonstrated that KRAS and BRAF mutations induce anoikis resistance in colon cancer (Caco‑2) cells. In 3D cultures, Caco‑2 cells transfected with mutated KRAS or BRAF formed multicellular structures analogous to anoikis‑resistant subpopulations in actual carcinomas, and serve as an in vitro model for anoikis resistance. Caco‑2 cell lines were constructed, with KRAS or BRAF mutations, using retroviral delivery. The current study investigated anoikis resistance using an Annexin V apoptosis test from suspension cultures. 3D in vitro cultures, which were generated in collagen‑matrigel mixtures, were assessed using confocal microscopy. 3D cultures embedded in paraffin were analyzed using conventional histopathology. In suspension cultures, Caco‑2 cells with KRAS or BRAF mutations indicated a significantly lower proportion of Annexin positivity than the native Caco‑2 cells, indicating that these mutations induce anoikis resistance in Caco‑2 cells. 3D cultures displayed native Caco‑2 cells forming polarized cysts with a single layer thick epithelium, whereas Caco‑2 cells with KRAS or BRAF mutations formed partially filled cystic structures or solid round structures where only the outermost layer was in contact with the ECM. Additionally, KRAS mutations induced reversed polarity to Caco‑2 cells along with the emergence of solid growth. The present study demonstrated that KRAS and BRAF mutations induce anoikis resistance in Caco‑2 colorectal cancer cells. The growth patterns generated from the KRAS and BRAF mutated cells in 3D cultures revealed a resemblance to the putative anoikis‑resistant subpopulations in actual carcinomas, including micropapillary structures and solid tumor cell islands. Additionally, KRAS mutation induced the emergence of inverted polarity. In conclusion, 3D cultures with modified Caco‑2 cells serve as a valid in vitro model for anoikis resistance and inverted polarity.

Dataset Information

Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells.

Publications

Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells.

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