Patankar, M., Eskelinen, S., Tuomisto, A., Mäkinen, M.J., & Karttunen, T.J. (2019). KRAS and BRAF mutations induce anoikis resistance and characteristic 3D phenotypes in Caco‑2 cells. Molecular Medicine Reports, 20, 4634-4644. https://doi.org/10.3892/mmr.2019.10693
KRAS and BRAF mutations induce anoikis resistance and characteristic 3D phenotypes in Caco‑2 cells
|Author:||Patankar, Madhura1,2; Eskelinen, Sinikka1; Tuomisto, Anne1,2;|
1Department of Pathology, Cancer and Translational Medicine Research Unit, University of Oulu, 90014 Oulu, Finland
2Medical Research Center Oulu, University of Oulu, 90014 Oulu
3Department of Pathology, Oulu University Hospital, 90029 Oulu, Finland
|Online Access:||PDF Full Text (PDF, 1 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe2020042219893
|Publish Date:|| 2020-04-22
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.
Molecular medicine reports
|Pages:||4634 - 4644|
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
The present study was funded by Finnish State Medical Research funding, allocated by the Oulu University Hospital, Finland.
© Patankar et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY 4.0].