Abstract
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.
Highlights
Colorectal cancer (CRC) is the third most common cancer worldwide [1]
The present study demonstrated that KRAS and BRAF mutations induce anoikis resistance in Caco‐2 colorectal cancer cells
We have recently identified tumor cell subpopulations, such as micropapillary structures (MIPs) in human colorectal carcinomas, which lack contact with the extracellular matrix (ECM) but still show a lower apoptosis rate, indicating that these structures represent an anoikis‐resistant subpopulation [22]
Summary
Colorectal cancer (CRC) is the third most common cancer worldwide [1]. In CRCs, 30‐50% carry KRAS mutations, most commonly in codon 12/13 [2], and 10‐15% carry BRAF V600E mutations [3]. Since the inner cells within such clusters might be devoid of extracellular matrix, unlike the outer cells, which rest in the semisolid 3D culture medium with ECM [31,32], such cells in 3D cultures could possibly serve as a model for the anoikis‐resistant subpopulation of carcinoma cells seen in actual human cancers. It is unknown whether KRAS or BRAF mutations induce anoikis resistance in Caco‐2 cells. We evaluated the role of Bim in the anoikis resistance of Caco‐2 cells
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