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A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

ABSTRACT: The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. Thus, significant efforts have been directed at identifying downstream targets on which RAS is dependent. These efforts have proven challenging, in part due to confounding factors such as reliance on two-dimensional adherent monolayer cell cultures that inadequately recapitulate the physiologic context to which cells are exposed in vivo. To overcome these issues, we implemented a high-throughput screening (HTS) approach using a spheroid-based 3-dimensional culture format, thought to more closely reflect conditions experienced by cells in vivo. Using isogenic cell pairs, differing in the status of KRAS, we identified Proscillaridin A as a selective inhibitor of cells harboring the oncogenic KRasG12V allele. Significantly, the identification of Proscillaridin A was facilitated by the 3D screening platform and would not have been discovered employing standard 2D culturing methods.

SUBMITTER: Kota S

PROVIDER: S-EPMC6138545 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

Kota Smitha S Hou Shurong S Guerrant William W Madoux Franck F Troutman Scott S Fernandez-Vega Virneliz V Alekseeva Nina N Madala Neeharika N Scampavia Louis L Kissil Joseph J Spicer Timothy P TP

Oncogene 20180510 32

The RAS proteins are the most frequently mutated oncogenes in cancer, with highest frequency found in pancreatic, lung, and colon tumors. Moreover, the activity of RAS is required for the proliferation and/or survival of these tumor cells and thus represents a high-value target for therapeutic development. Direct targeting of RAS has proven challenging for multiple reasons stemming from the biology of the protein, the complexity of downstream effector pathways and upstream regulatory networks. T ...[more]

PMID: 29743592

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Project description:Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Dataset Information

A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

Publications

A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype.

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